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The data is used in the paper "Count-based differential ....." by [http://www.nature.com/nprot/journal/v8/n9/abs/nprot.2013.099.html Anders et al 2013].
The data is used in the paper "Count-based differential ....." by [http://www.nature.com/nprot/journal/v8/n9/abs/nprot.2013.099.html Anders et al 2013]. The paper has been cited 8136 times according to scholar.google.com and 4330 times according to PubMed on Feb 7, 2019.


This page focuses on RNA-Seq. For DNA-Seq see the [[genome|Genome]] page.
This page focuses on RNA-Seq. For DNA-Seq see the [[genome|Genome]] page.
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Note
Note
* For alignment, [http://gettinggeneticsdone.blogspot.com/2012/11/star-ultrafast-universal-rna-seq-aligner.html STAR] is another choice.
* For alignment, [http://gettinggeneticsdone.blogspot.com/2012/11/star-ultrafast-universal-rna-seq-aligner.html STAR] is another choice.
* For trimmer, [http://www.usadellab.org/cms/index.php?page=trimmomatic trimmomatic] is another choice.
* For trimmer, [http://www.usadellab.org/cms/index.php?page=trimmomatic trimmomatic] is another choice. For example, we can use it to remove low quality reads (<30).
<syntaxhighlight lang='bash'>
java -jar trimmomatic-0.36.jar -phred33 -threads 8 file1.fastq.gz file2.fastq.gz -baseout file.fastq.gz AVGQUAL:30
 
java -jar trimmomatic-0.36.jar -phred33 -threads 8 file1.fastq.gz file2.fastq.gz -baseout file.fastq.gz HEADCROP:5 MINLEN:50 AVGQUAL:20
</syntaxhighlight>


== Installation ==
== Installation ==
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If we want to make these tools available for ALL users (ie system wide environment), we need to put these lines on '''/etc/profile''' file.
If we want to make these tools available for ALL users (ie system wide environment), we need to put these lines on '''/etc/profile''' file.


== HTSeq ==
== HTSeq and pysam ==
HTSeq requires Python 2 and pysam package. For pysam package, we have two ways to install it
HTSeq requires Python 2 and '''pysam''' package. For '''pysam''' package, we have two ways to install it


1. Use pip  
1. Use pip  
<syntaxhighlight lang='bash'>
<syntaxhighlight lang='bash'>
sudo apt-get install python-pip
sudo apt-get install python-pip
sudo -H pip install pysam
# sudo -H pip install pysam
sudo -H pip install pysam==0.10.0  # lock the version
</syntaxhighlight>
</syntaxhighlight>
The [http://stackoverflow.com/questions/28619686/what-is-the-h-flag-for-pip "-H"] flag is used to suppress a [[Python#warning:_Please_check_the_permissions_and_owner_of_that_directory|warning message]].
The [http://stackoverflow.com/questions/28619686/what-is-the-h-flag-for-pip "-H"] flag is used to suppress a [[Python#warning:_Please_check_the_permissions_and_owner_of_that_directory|warning message]].
The latest version of pysam may fail so it is not good to use the command '''pip install pysam'''.
<syntaxhighlight lang='bash'>
$ python
>>> import pysam
Traceback (most recent call last):
  File "<stdin>", line 1, in <module>
  File "/Library/Python/2.7/site-packages/pysam/__init__.py", line 5, in <module>
    from pysam.libchtslib import *
  File "pysam/libchtslib.pyx", line 1, in init pysam.libchtslib (pysam/libchtslib.c:12515)
ImportError: dlopen(/Library/Python/2.7/site-packages/pysam/libcutils.so, 2): Library not loaded: @rpath/pysam-0.11.2.2-py2.7-macosx-10.12-intel.egg/pysam/libcsamtools.so
  Referenced from: /Library/Python/2.7/site-packages/pysam/libcutils.so
  Reason: image not found
</syntaxhighlight>


2. Use [https://www2.warwick.ac.uk/fac/sci/systemsbiology/staff/dyer/software/htseq/ python to build/install].
2. Use [https://www2.warwick.ac.uk/fac/sci/systemsbiology/staff/dyer/software/htseq/ python to build/install].
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It would be interesting to monitor the disk space usage before, during and after the execution.
It would be interesting to monitor the disk space usage before, during and after the execution.
Other pipelines:
* [https://link.springer.com/protocol/10.1007%2F978-1-4939-7286-9_23 RNA-Seq Data Analysis: From Raw Data Quality Control to Differential Expression Analysis], Sep 2017. Bash and R scripts are included.
== Download example data (22 files in SRA format) ==
== Download example data (22 files in SRA format) ==


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== Convert SRA to FASTQ (sra -> fastq) ==
== Convert SRA to FASTQ (sra -> fastq) ==
(Update) [https://github.com/ncbi/sra-tools/wiki/HowTo:-fasterq-dump fasterq-dump] command. Note there is no '''--gzip''' option as in [https://ncbi.github.io/sra-tools/fastq-dump.html fastq-dump]. Use '''gzip''' to do that.
This takes a long time and it is not to run in parallel by default. At the end of execution, there are 30 fastq files because 8 SRA file are pair end.
This takes a long time and it is not to run in parallel by default. At the end of execution, there are 30 fastq files because 8 SRA file are pair end.
<pre>
<pre>
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-rw-r--r-- 1 brb brb 3.2G Feb 10 14:07 SRR031729.fastq
-rw-r--r-- 1 brb brb 3.2G Feb 10 14:07 SRR031729.fastq
</pre>
</pre>
=== FASTQ PAIR ===
[https://github.com/linsalrob/fastq-pair FASTQ PAIR] Rewrite paired end fastq files to make sure that all reads have a mate and to separate out singletons


== Access quality control through ShortRead or [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ FastQC] ==
== Access quality control through ShortRead or [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ FastQC] ==
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# [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/3%20Analysis%20Modules/9%20Overrepresented%20Sequences.html Overpresented sequences]
# [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/3%20Analysis%20Modules/9%20Overrepresented%20Sequences.html Overpresented sequences]
# [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/3%20Analysis%20Modules/11%20Kmer%20Content.html Kmer Content]
# [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/3%20Analysis%20Modules/11%20Kmer%20Content.html Kmer Content]
=== Encoding ===
* [https://en.wikipedia.org/wiki/FASTQ_format#Encoding Encoding] Starting in Illumina 1.8, the quality scores have basically returned to the use of the Sanger format (Phred+33).
* [http://seqanswers.com/forums/showthread.php?t=8002 all the FASTQ sequences from ncbi are Phred+33] (same as Sanger).


== Download reference genome (for Tophat) ==
== Download reference genome (for Tophat) ==
Line 708: Line 741:
</pre>
</pre>
where each line consists of '''chromosome, 1-based coordinate, reference base, the number of reads covering the site, read bases''' and '''base qualities'''.
where each line consists of '''chromosome, 1-based coordinate, reference base, the number of reads covering the site, read bases''' and '''base qualities'''.
Or use igvtools https://hpc.nih.gov/apps/IGV.html


== Inspect alignments with [http://www.broadinstitute.org/igv/ IGV] ==
== Inspect alignments with [http://www.broadinstitute.org/igv/ IGV] ==
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* The memory handling is not as good as IGV (tested using Anders2013 data with the same 4000M setting).
* The memory handling is not as good as IGV (tested using Anders2013 data with the same 4000M setting).
* There are several tutorial videos for IGB and the user guide is good.
* There are several tutorial videos for IGB and the user guide is good.
=== Other visualization tools ===
[http://www.sthda.com/english/wiki/genomics CHAPTER V : Visualizing next generation sequencing data]


== Count reads using htseq-count (sam -> count) ==
== Count reads using htseq-count (sam -> count) ==
Line 1,014: Line 1,052:
* can produce the same result as htseq
* can produce the same result as htseq
* java based
* java based
=== featureCount from Subread ===
* [http://subread.sourceforge.net/ Subread]: high-performance read alignment, quantification and mutation discovery
* [http://bioinf.wehi.edu.au/featureCounts/ featureCounts]: a ultrafast and accurate read summarization program


== DESeq2 normalization ==
== DESeq2 normalization ==
* https://support.bioconductor.org/p/66067/
* [https://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html Analyzing RNA-seq data with DESeq2]
* [https://www.jianshu.com/p/2689e9a1d10c DESeq2详细用法]
* https://github.com/mgonzalezporta/TeachingMaterial/blob/master/doc/25.normalising.md
* https://github.com/mgonzalezporta/TeachingMaterial/blob/master/doc/25.normalising.md
* http://www.sthda.com/english/wiki/rna-sequencing-data-analysis-counting-normalization-and-differential-expression
* http://www.sthda.com/english/wiki/rna-sequencing-data-analysis-counting-normalization-and-differential-expression
* '''[https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0550-8 Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2]''' by Love, Huber and Anders, 2014. Keywords: LFC (logarithmic fold change), shrinkage for dispersion estimation, MAP (maximum a posteriori), Shrinkage estimation of logarithmic fold changes, Wald test, Cook's distance for outlier detection. R packages: DESeq2, '''GenomicRanges''' and '''GenomicAlignments'''.  
* '''[https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0550-8 Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2]''' by Love, Huber and Anders, 2014. Keywords: LFC (logarithmic fold change), shrinkage for dispersion estimation, MAP (maximum a posteriori), Shrinkage estimation of logarithmic fold changes, Wald test, Cook's distance for outlier detection. R packages: DESeq2, '''GenomicRanges''' and '''GenomicAlignments'''.  
<syntaxhighlight lang='rsplus'>
: <syntaxhighlight lang='rsplus'>
install.packages("~/Downloads/DESeq2paper_1.3.tar.gz", repos = NULL, type="source")
install.packages("~/Downloads/DESeq2paper_1.3.tar.gz", repos = NULL, type="source")
source("http://bioconductor.org/biocLite.R") # https:// not work on my R 3.2.5 Ubuntu
source("http://bioconductor.org/biocLite.R") # https:// not work on my R 3.2.5 Ubuntu
Line 1,037: Line 1,080:
</syntaxhighlight>
</syntaxhighlight>
* The count matrix and metadata, including the gene model and sample information, are stored in an S4 class derived from the SummarizedExperiment class of the GenomicRanges package. SummarizedExperiment objects containing count matrices can be easily generated using the '''summarizeOverlaps()''' function of the GenomicAlignments package.  
* The count matrix and metadata, including the gene model and sample information, are stored in an S4 class derived from the SummarizedExperiment class of the GenomicRanges package. SummarizedExperiment objects containing count matrices can be easily generated using the '''summarizeOverlaps()''' function of the GenomicAlignments package.  
* [http://genomebiology.biomedcentral.com/articles/10.1186/gb-2010-11-10-r106 Differential expression analysis for sequence count data] for DESeq package from Anders & Huber, 2010.
* [https://youtu.be/mq6UvDneKc0?t=746 Log2 fold-change & DESeq2 model in a nutshell] (youtube)
 
=== Input ===
<syntaxhighlight lang='rsplus'>
DESeqDataSetFromHTSeqCount(sampleTable, directory, design)
# sampleTable is like colData in the next 2 functions
 
DESeqDataSetFromTximport(txi, colData, design)
# txi is obtained by tximport()
 
DESeqDataSetFromMatrix(countData, colData, design)
# count data input
 
DESeqDataSet(se, design)
# e.g. the ''SummarizedExperiment'' object created by tximeta()
# 'airway' package/object is an example
</syntaxhighlight>
 
==== collapseReplicates ====
* [https://youtu.be/gKnfP2_Xdpo StatQuest: RNA-seq - the problem with technical replicates]
* [https://support.bioconductor.org/p/9152326/ Accounting for technical replicates in DESeq2]
<syntaxhighlight lang='rsplus'>
<syntaxhighlight lang='rsplus'>
dds <- makeExampleDESeqDataSet(m=12)
# make data with two technical replicates for three samples
dds$sample <- factor(sample(paste0("sample",rep(1:9, c(2,1,1,2,1,1,2,1,1)))))
dds$run <- paste0("run",1:12)
ddsColl <- collapseReplicates(dds, dds$sample, dds$run)
# dds$sample is a grouping variable
# examine the colData and column names of the collapsed data
colData(ddsColl)
colnames(ddsColl)
# DataFrame with 9 rows and 4 columns
#        condition  sample        run runsCollapsed
#          <factor> <factor> <character>  <character>
# sample1        A  sample1        run6    run6,run9
# sample2        B  sample2      run12        run12
# sample3        A  sample3        run2          run2
# sample4        A  sample4        run5    run5,run7
# sample5        A  sample5        run1          run1
# sample6        A  sample6        run4          run4
# sample7        B  sample7        run8    run8,run10
# sample8        A  sample8        run3          run3
# sample9        B  sample9      run11        run11
# check that the sum of the counts for "sample1" is the same
# as the counts in the "sample1" column in ddsColl
matchFirstLevel <- dds$sample == levels(dds$sample)[1]
stopifnot(all(rowSums(counts(dds[,matchFirstLevel])) == counts(ddsColl[,1])))
</syntaxhighlight>
=== Normalized counts = raw counts/sizeFactor ===
<ul>
<li>[https://support.bioconductor.org/p/66067/ How to access the normalized data of a DESeqDataSet]
<li>How exactly DESeq2 correct for differences in sequencing depth and library composition to get normalized counts?
* To normalize for sequencing depth and RNA composition, DESeq2 uses the '''median of ratios''' method. On the user-end there is only one step, but on the back-end there are multiple steps involved. The algorithm performs multiple steps such as computing the '''pseudo-reference''': geometric mean for each gene across all samples.
<li>[https://statquest.org/statquest-deseq2-part-1-library-normalization/ StatQuest: DESeq2 part 1: Library Normalization] (480p only)
# take the log of all the values (log0 = -Inf)
# average each row
# filter out genes with infinity
# subtract the average log value from the log(counts)
# calculate the median of the ratios for each sample
# convert the medians to "normal numbers" to get the final scaling factors for each sample (exp(these medians))
# divide the original read counts by the scaling factors
<li>Summary
# '''Calculate the normalization factor (size factor) for each sample''':
## '''Create a pseudo-reference sample''': For each gene, calculate the geometric mean of counts across all samples. This geometric mean serves as a pseudo-reference sample.
## '''Calculate the ratio of each sample to the reference''': For each gene in each sample, divide the count by the geometric mean (pseudo-reference) calculated in step 1. This gives the '''ratio''' of each sample to the reference.
## For each sample, calculate the median of the ratios obtained in step 2. This median value is the '''size factor''' for that sample.
# '''Calculate the normalized count values''': Divide the raw counts for each gene in each sample by the size factor for that sample. This gives the normalized count values.
<li> [http://genomebiology.biomedcentral.com/articles/10.1186/gb-2010-11-10-r106 Differential expression analysis for sequence count data] for DESeq package from Anders & Huber, 2010. If we want normalized counts, we need to run 1) '''estimateSizeFactors()''' for the normalization, and 2) '''counts(dds, normalized = TRUE)''' to extract normalized counts or '''normTransform(dds)''' on log2 scale. The normalization is part of the DESeq() function so you can extract counts after running that as well.
<syntaxhighlight lang='r'>
source("http://bioconductor.org/biocLite.R"); biocLite("pasilla")
source("http://bioconductor.org/biocLite.R"); biocLite("pasilla")
directory <- system.file("extdata", package="pasilla", mustWork=TRUE)
directory <- system.file("extdata", package="pasilla", mustWork=TRUE)
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# FBgn0000008:008      2.7260184      1.246732      0.000000        0.0000000        0.6178182
# FBgn0000008:008      2.7260184      1.246732      0.000000        0.0000000        0.6178182
</syntaxhighlight>
</syntaxhighlight>
<pre>
# Retrieve raw counts and size factors
raw_counts <- counts(dds)
size_factors <- sizeFactors(dds)


=== Negative binomial regression for count data ===
# Manually calculate normalized counts
http://statistics.ats.ucla.edu/stat/r/dae/nbreg.htm
manual_normalized_counts <- sweep(raw_counts, 2, size_factors, FUN = "/")


== DESeq2 for DE analysis ==
# Retrieve normalized counts from DESeq2
<syntaxhighlight lang='rsplus'>
normalized_counts <- counts(dds, normalized = TRUE)
> samplesDESeq = with(samples, data.frame(
        shortname = I(shortname),
        countf = I(countf),
        condition = condition,
        LibraryLayout = LibraryLayout))


> samplesDESeq
# Compare the two matrices
all.equal(manual_normalized_counts, normalized_counts)
</pre>
</ul>


shortname              countf condition LibraryLayout
=== High-Throughput Count Data ===
1  CT.PA.1  Untreated-3.count      CTL        PAIRED
http://web.stanford.edu/class/bios221/book/Chap-CountData.html
2  CT.PA.2  Untreated-4.count      CTL        PAIRED
3  KD.PA.3 CG8144_RNAi-3.count        KD        PAIRED
4  KD.PA.4 CG8144_RNAi-4.count        KD        PAIRED
5  CT.SI.5  Untreated-1.count      CTL        SINGLE
6  KD.SI.6 CG8144_RNAi-1.count        KD        SINGLE
7  CT.SI.7  Untreated-6.count      CTL        SINGLE


> library("DESeq")
=== estimateSizeFactorsForMatrix() ===
cds <- newCountDataSetFromHTSeqCount(samplesDESeq) # quick, 15682 features, 7 samples
We can compare the output of estimateSizeFactorsForMatrix() with what you get by simply taking the column sums. See [https://www.huber.embl.de/msmb/Chap-CountData.html High-Throughput Count Data].
cds <- estimateSizeFactors(cds) # quick
sizeFactors(cds)


#  CT.PA.1  CT.PA.2  KD.PA.3  KD.PA.4  CT.SI.5  KD.SI.6  CT.SI.7
Use a '''robust regression''' to estimate the size factor instead of taking the sum of reads per sample.
# 0.6991612 0.8104921 0.8217403 0.8941097 1.6431467 1.3720872 1.1041186


cds <- estimateDispersions(cds) # quick
The output estimateSizeFactorsForMatrix() is a vector of the number of samples.
res <- nbinomTest(cds,"CTL","KD") # 44 seconds
sum(res$pval <= .05, na.rm=T) # [1] 1574
sum(res$padj <= .05, na.rm=T) # [1] 730


options(width=100)
=== estimateDispersions() ===
res[1:3,]
[https://twitter.com/vitaliikl/status/1681754077296828416 NB dispersion in DESeq2 correspond to Gamma-Poisson parametrisation?]
#           id baseMean baseMeanA baseMeanB foldChange log2FoldChange      pval padj
<pre>
# 1 FBgn0000003  0.000000  0.000000  0.000000        NaN            NaN        NA  NA
dds <- makeExampleDESeqDataSet() # 1000 x 12
# 2 FBgn0000008 88.526695 88.486694 88.580029  1.001055    0.001520941 0.9298627    1
dds <- estimateSizeFactors(dds)
# 3 FBgn0000014  3.054418  2.327533  4.023599  1.728697    0.789684778 0.6839858    1
dds <- estimateDispersions(dds)
</syntaxhighlight>
length(dispersions(dds)) # [1] 1000
 
plotDispEsts(dds) # x=mean of normalized counts, y=dispersion
# plots the per-gene dispersion estimates together with
# the fitted mean-dispersion relationship
</pre>
So the output of estimateDispersions() is a vector of the number of genes.
 
* [https://youtu.be/ZyRT1z1Sz3g?t=34 Two factors that we use to decide if we use shrinkage]: '''high dispersion values, low gene counts'''
* [https://youtu.be/ZyRT1z1Sz3g?t=362 Estimating Dispersion]


= File Format =
=== DESeq() and gene filtering ===
* http://genome.ucsc.edu/FAQ/FAQformat.html which includes a lot of common formats like BAM, BED, bedGraph, bigBed, bigWig, VCF, WIG, et al.
* [https://support.bioconductor.org/p/79855/#79856 Remove genes with very low counts for all samples or let DESeq2 perform independent filtering?]  The safest threshold would be to not filter anything above row sum of 0...  And our recommendation (and the default in DESeq2) is to let the '''genefilter''' software optimize the threshold, such that sensitivity (statistical power) is maximized.
* http://stephenturner.us/biofiletypes/  
* [https://www.rdocumentation.org/packages/DESeq2/versions/1.12.3/topics/plotMA plotMA()], [https://www.rdocumentation.org/packages/DESeq2/versions/1.12.3/topics/plotCounts plotCounts]
* [https://support.bioconductor.org/p/9151218/ DESeq: low p-values for genes with zero counts in all but one sample of the compared groups]


== [http://en.wikipedia.org/wiki/FASTQ_format fastq] ==
=== Combine two DESeq2 objects (dds) for analysis ===
[https://support.bioconductor.org/p/9150577/ How to combine two DESeq2 objects (dds) for analysis]


The [http://en.wikipedia.org/wiki/FASTQ_format#Format_converters wikipedia] website provides information to convert FASTQ to FASTA format (when do we want to do that?).
=== Designs and contrasts ===
* [https://www.atakanekiz.com/technical/a-guide-to-designs-and-contrasts-in-DESeq2/ A guide to designs and contrasts in DESeq2]
* [https://support.bioconductor.org/p/9159215/ How to correct for a covariate in DESeq2 that is linear with the condition of interest]. Nested design.
* [https://support.bioconductor.org/p/105016/ DESeq2 experimental design, 2 controls and 2 treated]. Goal is to see how/if the two cell types respond differently to treatment.
** '''~celltype + celltype:treatment '''
* [https://support.bioconductor.org/p/9159392/ Deseq2 design formula and analysis with internal controls]. Goal is to compare two groups by taking the treatment into consideration.
** Factor1: group/cell-type
** Factor2: treatment


=== fastq format ===
=== results(contrast) ===
A FASTQ file normally uses four lines per sequence.
<ul>
<pre>
<li>[https://rdocumentation.org/packages/DESeq2/versions/1.12.3/topics/results ?results]
@SEQ_ID
<li>Syntax: contrast=c('variable', 'numerator', 'denominator'). Example: contrast=c('Trt', 'Yes', 'No')
GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT
<li>Example 1: two conditions
+
<syntaxhighlight lang='r'>
!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65
dds <- makeExampleDESeqDataSet(m=4)
</pre>
* Line 1 begins with a '@' character and is followed by a sequence identifier and an optional description (like a FASTA title line).
* Line 2 is the raw sequence letters.
* Line 3 begins with a '+' character and is optionally followed by the same sequence identifier (and any description) again.
* Line 4 encodes the quality values for the sequence in Line 2, and must contain the same number of symbols as letters in the sequence.


It seems line 3 & 4 are not required. See the simulated fastq files on http://genomespot.blogspot.com/2014/11/dna-aligner-accuracy-bwa-bowtie-soap.html. I can use BWA to run alignment without a problem:)
dds <- DESeq(dds)
res <- results(dds, contrast=c("condition","B","A"))  # mean_B - mean_A
res
#log2 fold change (MLE): condition B vs A
#Wald test p-value: condition B vs A
#DataFrame with 1000 rows and 6 columns
#          baseMean log2FoldChange    lfcSE      stat    pvalue      padj
#        <numeric>      <numeric> <numeric> <numeric> <numeric> <numeric>
#gene1      2.18621      -3.029859  2.910105 -1.041151  0.297806  0.999351
#gene2    14.97050      -0.850521  1.112673 -0.764395  0.444632  0.999351
#gene3      9.31174      0.448988  1.379610  0.325446  0.744844  0.999351
#gene4    84.12136      0.333454  0.593804  0.561556  0.574419  0.999351
#gene5    21.52635      -0.538152  1.025702 -0.524667  0.599815  0.999351


=== Paired end ===
counts(dds, normalized = T)[1:3, ]
[http://www.cureffi.org/2012/12/19/forward-and-reverse-reads-in-paired-end-sequencing/ Forward and reverse reads in paired-end sequencing]
#        sample1    sample2  sample3  sample4
#gene1  6.812072  0.9935038  0.00000 0.9392723
#gene2 11.677838 26.8246037 15.74392 5.6356338
#gene3  7.785225  7.9480307 18.69590 2.8178169


The paired files can be two formats. See [https://bioinf.comav.upv.es/courses/sequence_analysis/sequence_file_formats.html. Bioinformatics at COMAV]
colData(dds)
#DataFrame with 4 rows and 2 columns
#        condition sizeFactor
#        <factor>  <numeric>
#sample1        A    1.02759
#sample2        A    1.00654
#sample3        B    1.01627
#sample4        B    1.06465


Format1:
# gene 1
<pre>
> mean(c(6.81, .99)) # condition A
Fastq file 1
[1] 3.9
@molecule_1 1st_read_from_pair
> mean(c(0, 0.939))  # condition B
@molecule_2 1st_read_from_pair
[1] 0.4695
@molecule_3 1st_read_from_pair
</syntaxhighlight>
 
<li>Example 2: two conditions, one continuous variable (age)
<syntaxhighlight lang='r'>
set.seed(1234)
dds <- makeExampleDESeqDataSet(n=100,m=12)
dds$age <- rpois(12, 50)
design(dds) <- ~ condition + age
dds <- DESeq(dds)
resultsNames(dds)
# [1] "Intercept"        "condition_B_vs_A" "age"
 
results(dds)  # OR  results(dds, name="age")
#log2 fold change (MLE): age
#Wald test p-value: age
#DataFrame with 100 rows and 6 columns
#        baseMean log2FoldChange    lfcSE      stat    pvalue      padj
#        <numeric>      <numeric> <numeric>  <numeric> <numeric> <numeric>
#gene1    2.022210    -0.04620020 0.1445006 -0.3197232  0.749178  0.920829
#gene2  24.571867    0.00360519 0.0484505  0.0744097  0.940684  0.991664
#gene3  82.491501    0.00153923 0.0329532  0.0467095  0.962745  0.991664
 
results(dds, contrast=c("condition","B","A"))
# OR results(dds, name="condition_B_vs_A")


Fastq file 2
#log2 fold change (MLE): condition B vs A
@molecule_1 2nd_read_from_pair
#Wald test p-value: condition B vs A
@molecule_2 2nd_read_from_pair
#DataFrame with 100 rows and 6 columns
@molecule_3 2nd_read_from_pair
#        baseMean log2FoldChange    lfcSE      stat    pvalue      padj
</pre>
#        <numeric>      <numeric> <numeric> <numeric> <numeric> <numeric>
Format 2:
#gene1    2.022210      3.0976656  1.854100  1.670711 0.0947788  0.910079
<pre>
#gene2  24.571867    -0.1667077  0.635550 -0.262305 0.7930867  0.982211
Interleaved Fastq file
#gene3  82.491501      0.2188290  0.431659  0.506948 0.6121910  0.982211
@molecule_1 1st_read_from_pair
</syntaxhighlight>
@molecule_1 2nd_read_from_pair
@molecule_2 1st_read_from_pair
@molecule_2 2nd_read_from_pair
@molecule_3 1st_read_from_pair
@molecule_3 2nd_read_from_pair
</pre>


Note from my experiment, if we flip the two fastq files order in the BWA command, the resulting bam file will diff at FLAG (2nd) column. The final vcf file is not changed.
<li>Example 3: two conditions, two genotypes, with an interaction term
<syntaxhighlight lang='r'>
set.seed(1234)
dds <- makeExampleDESeqDataSet(n=100,m=12)
dds$genotype <- factor(rep(rep(c("I","II"),each=3),2))


=== FastQValidator ===
design(dds) <- ~ genotype + condition + genotype:condition
http://genome.sph.umich.edu/wiki/FastQValidator
dds <- DESeq(dds)
resultsNames(dds)
# [1] "Intercept"            "genotype_II_vs_I"   
# [3] "condition_B_vs_A"      "genotypeII.conditionB"


== [http://en.wikipedia.org/wiki/FASTA_format fasta] ==
# the condition effect for genotype I (the main effect)
results(dds, contrast=c("condition","B","A"))


FASTA format is a text-based format for representing nucleotide sequences . cf. FASTQ files provide more information than FASTA since FASTQ format stores both a biological sequence (usually nucleotide sequence) and its corresponding quality scores.
# the condition effect for genotype II
# this is, by definition, the main effect *plus* the interaction term
# (the extra condition effect in genotype II compared to genotype I).
results(dds, list( c("condition_B_vs_A","genotypeII.conditionB") ))


The reference genome is saved in FASTA format.
# the interaction term, answering: is the condition effect *different* across genotypes?
results(dds, name="genotypeII.conditionB")
</syntaxhighlight>
</ul>


Normally the first line in a FASTA file starts with a ">" (greater-than) symbol. Subsequent lines starting with a semicolon would be ignored by software. Following the initial line (used for a unique description of the sequence) is the actual sequence itself in standard one-letter code. A multiple sequence FASTA format would be obtained by concatenating several single sequence FASTA files.  
=== results(alpha, independentFiltering) ===
By default alpha=.1, Set independentFiltering=FALSE if we don't like independent filtering.


For the fruit fly genome file ''Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa''. The '''grep -n''' command can be used to get the row numbers of some keyword (chromosome names in this case).
=== Negative binomial regression and returned from results() ===
A matrix with columns:
<ul>
<li>'''baseMean''': mean of normalized counts of all samples, normalizing for sequencing depth. It does not take into account gene length. The base mean is used in DESeq2 only for estimating the dispersion of a gene. See [https://support.bioconductor.org/p/75244/#75245 DESeq2 baseMean counts].  
<pre>
<pre>
$ head Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa
rowMeans(counts(dds, normalized=TRUE))
>2L dna:chromosome chromosome:BDGP5:2L:1:23011544:1 REF
</pre>
CGACAATGCACGACAGAGGAAGCAGAACAGATATTTAGATTGCCTCTCATTTTCTCTCCC
</li>
ATATTATAGGGAGAAATATGATCGCGTATGCGAGAGTAGTGCCAACATATTGTGCTCTTT
<li>'''log2FoldChange''' [https://support.bioconductor.org/p/77021/ log2FoldChange calculation in DESeq2 output], [https://youtu.be/o288b1upip8?t=572 2019 STAT115 Lect7.4 DESeq2] by Shirley Liu. [https://youtu.be/mq6UvDneKc0?t=608 Theory behind DESeq2] & [https://youtu.be/mq6UvDneKc0?t=920 Null hypothesis]. [https://hbctraining.github.io/DGE_workshop/lessons/05_DGE_DESeq2_analysis2.html Introduction to DGE] from hbctraining.
GATTTTTTGGCAACCCAAAATGGTGGCGGATGAACGAGATGATAATATATTCAAGTTGCC
: <math>
GCTAATCAGAAATAAATTCATTGCAACGTTAAATACAGCACAATATATGATCGCGTATGC
\begin{align}
GAGAGTAGTGCCAACATATTGTGCTAATGAGTGCCTCTCGTTCTCTGTCTTATATTACCG
K_{ij} & \sim NB(\mu_{ij}, \alpha_i)  \\
CAAACCCAAAAAGACAATACACGACAGAGAGAGAGAGCAGCGGAGATATTTAGATTGCCT
\mu_{ij} &= s_{j} q_{ij} \\
ATTAAATATGATCGCGTATGCGAGAGTAGTGCCAACATATTGTGCTCTCTATATAATGAC
\log_2(q_{ij}) &= x_{j} \beta_i
TGCCTCTCATTCTGTCTTATTTTACCGCAAACCCAAATCGACAATGCACGACAGAGGAAG
\end{align}
CAGAACAGATATTTAGATTGCCTCTCATTTTCTCTCCCATATTATAGGGAGAAATATGAT
</math>
<math>s_j</math> is the '''size factor''' for sample 𝑗, accounting for differences in sequencing depth. </BR>
<math>q_{ij}</math> is the '''normalized mean count''' for gene 𝑖 in sample 𝑗. </BR>
<span style="color: red">The coefficients <math>\beta_i</math> estimate give the log2 fold changes </span> for gene ''i'' for each column of the '''model matrix''' <math>X</math>.
* In a simple linear regression <math> y = \beta_0 + \beta_1 x + \epsilon</math>, if the covariate X takes only 0 or 1, we can show that the slope estimate <math>\frac{\sum(x_i-\bar{x})(y_i-\bar{y})}{\sum(x_i -\bar{x})^2}</math> can be simplified to the difference of group means <math>\bar{y}_1 - \bar{y}_0</math>. This matches the idea of the fold change in gene expression.
* You can obtain standard log fold changes (no shrinkage) by using: DESeq(dds, betaPrior=FALSE)
</li>
 
<li>The null hypothesis being tested is: <math>H_0: \beta_i = 0</math>. This tests whether the log fold change for the condition is zero, meaning there is no differential expression for gene 𝑖 between conditions.


$ grep -n ">" Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa
<li>'''lfcSE''' [https://support.bioconductor.org/p/93559/ standard error value (lfcSE) returned by DeSeq2]     
1:>2L dna:chromosome chromosome:BDGP5:2L:1:23011544:1 REF
<li>[https://support.bioconductor.org/p/113280/ Why the logFC is different between edgeR,DESeq2 results and fpkm logFC(log2(FPKM_tumor_mean/FPKM_normal_mean)?] LogFCs are computed by negative binomial generalized linear models.
383528:>2LHet dna:chromosome chromosome:BDGP5:2LHet:1:368872:1 REF
</li>
389677:>2R dna:chromosome chromosome:BDGP5:2R:1:21146708:1 REF
<li>[https://support.bioconductor.org/p/9151066/ Why my log2fcs are too high?] </li>
742124:>2RHet dna:chromosome chromosome:BDGP5:2RHet:1:3288761:1 REF
<li> stat: negative binomial GLM fitting for βi and Wald statistics by nbinomWaldTest(). log2Foldchange除以标准差所得
796938:>3L dna:chromosome chromosome:BDGP5:3L:1:24543557:1 REF
</li>    
1205999:>3LHet dna:chromosome chromosome:BDGP5:3LHet:1:2555491:1 REF
<li>pvalue & padj ([https://hbctraining.github.io/DGE_workshop_salmon_online/lessons/05b_wald_test_results.html Exploring DESeq2 results: Wald test] from hbctraining)
1248592:>3R dna:chromosome chromosome:BDGP5:3R:1:27905053:1 REF
</li>
1713678:>3RHet dna:chromosome chromosome:BDGP5:3RHet:1:2517507:1 REF
</ul>
1755638:>4 dna:chromosome chromosome:BDGP5:4:1:1351857:1 REF
1778170:>U dna:chromosome chromosome:BDGP5:U:1:10049037:1 REF
1945655:>Uextra dna:chromosome chromosome:BDGP5:Uextra:1:29004656:1 REF
2429067:>X dna:chromosome chromosome:BDGP5:X:1:22422827:1 REF
2802782:>XHet dna:chromosome chromosome:BDGP5:XHet:1:204112:1 REF
2806185:>YHet dna:chromosome chromosome:BDGP5:YHet:1:347038:1 REF
2811970:>dmel_mitochondrion_genome dna:chromosome chromosome:BDGP5:dmel_mitochondrion_genome:1:19517:1 REF


$ wc -l *.fa
=== Fold-change with continuous variables ===
2812296 Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa
[https://support.bioconductor.org/p/9159370/ how limma work with continous variables]. It's the slope/fitted coefficient of the line.
</pre>


For human,
=== Normalization-based vs log-ratio transformation-based methods ===
* [https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-018-2261-8 Benchmarking differential expression analysis tools for RNA-Seq: normalization-based vs. log-ratio transformation-based methods]
* If we use log2(normalized count + 1)，有很低counts的基因将倾向于主导结果。作为一种解决方案，DESeq2为counts数据提供了stabilize the variance across the mean的转换。其中之一是regularized-logarithm transformation or rlog2。对于counts较高的基因，rlog转换可以得到与普通log2转换相似的结果。然而，对于counts较低的基因，所有样本的值都缩小到基因的平均值。See [https://www.jianshu.com/p/2689e9a1d10c DESeq2详细用法] and the section [http://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#data-transformations-and-visualization Data transformations and visualization] from the DESeq2 vignette.
 
=== Error with DESeq2 ===
[https://help.galaxyproject.org/t/error-with-deseq2-every-gene-contains-at-least-one-zero/564 Error with DESeq2 “every gene contains at least one zero”]
 
=== Source code ===
* counts(), normalizationFactors(), `normalizationFactor<-`, estimateSizeFactors(). [https://github.com/mikelove/DESeq2 DESeq2] -> R -> [https://github.com/mikelove/DESeq2/blob/master/R/methods.R methods.R]
* [https://bioinformatics.stackexchange.com/a/201 How can I extract normalized read count values from DESeq2 results?]
<pre>
<pre>
$ grep -n ">" ~/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa
? normalizationFactors
1:>chrM
dds <- makeExampleDESeqDataSet(n=100, m=4)
334:>chr1
 
4985348:>chr2
normFactors <- matrix(runif(nrow(dds)*ncol(dds),0.5,1.5),
9849337:>chr3
                      ncol=ncol(dds),nrow=nrow(dds),
13809787:>chr4
                      dimnames=list(1:nrow(dds),1:ncol(dds)))
17632874:>chr5
21251181:>chr6
24673484:>chr7
27856259:>chr8
30783541:>chr9
33607811:>chr10
36318507:>chr11
39018639:>chr12
41695678:>chr13
43999077:>chr14
46146069:>chr15
48196698:>chr16
50003795:>chr17
51627701:>chr18
53189247:>chr19
54371828:>chr20
55632340:>chr21
56594939:>chr22
57621032:>chrX
60726445:>chrY
</pre>


=== chromosome names ===
normFactors <- normFactors / exp(rowMeans(log(normFactors)))
When I apply the above method to examine the genome.fa files from Ensembl, NCBI and UCSC using the command
normalizationFactors(dds) <- normFactors
<syntaxhighlight lang='bash'>
 
grep -n ">" genome.fa | cut -d' ' -f1 | cut -d '>' -f2
dds <- DESeq(dds)
# for example
res = results(dds)
grep -n ">" ~/Downloads/Homo_sapiens.GRCh38.dna.chromosome.22.fa | cut -d' ' -f1 | cut -d '>' -f2 
res[order(res$padj), ] %>% head
# 22
 
</syntaxhighlight>
class(dds)
I get the following result
showMethods(class="DESeqDataSet") # a long list
# Ensembl:
 
## GRCh37: 1, 2, 3, ...., X, Y, MT
isS4(assays(dds))    # [1] TRUE
## GRCh38: 1, 2, 3, ... (see note below)
slotNames(assays(dds))
# NCBI:
# [1] "listData"        "elementType"    "elementMetadata" "metadata"     
## build37.1, build37.2: 1, 2, 3, ....
names(assays(dds))
## GRCh38: chr1, chr2, ...., chr22, chrX, chrY, chrM, chr1_KI270706v1_random, ....., chrEBV
# [1] "counts"              "normalizationFactors" "mu"  "H"   
# UCSC:
## hg18, hg19, hg38: chr1, chr2, ...., chr22, chrX, chrY, chrM


Note that
all(counts(dds) / normFactors == counts(dds, norm=T)) # TRUE
* <span style="color: red">Neither Illumina or Biowulf provide GRCh38 from Ensembl</span> (maybe for a good reason). Ensembl (http://ftp.ensembl.org/pub/release-76/fasta/homo_sapiens/dna) does provide GRCh38.
</pre>
* GRCh38 from Ensembl is still following its GRCh37 convention; it won't include 'chr' on their chromosome names. That is, even the same name GRCh38 was used in both Ensembl and NCBI, they have different conventions.


=== chromosome sizes ===
=== DESeq normalization 2010 ("median ratio") ===
https://www.biostars.org/p/134807/
* [https://twitter.com/mikelove/status/1472174438372319233 DESeq normalization 2010 ("median ratio") ]
* [https://genomebiology.biomedcentral.com/articles/10.1186/gb-2010-11-10-r106 2010 Paper]
* [https://rdrr.io/bioc/alpine/man/normalizeDESeq.html normalizeDESeq(mat, cutoff)]


<syntaxhighlight lang='bash'>
== edgeR normalization ==
GENOMEFA=Arabidopsis_thaliana.TAIR10.23.dna.genome.fa
* [https://web.stanford.edu/class/bios221/labs/rnaseq/lab_4_rnaseq.html RNA Sequence Analysis in R: edgeR]
<pre>
library(baySeq)
data(mobData)
head(mobData)
data(mobAnnotation)
#?mobAnnotation
head(mobAnnotation)
library(edgeR)
d <- DGEList(counts=mobData,group=factor(mobDataGroups))
d
d.full <- d # keep the old one in case we mess up
head(d.full$counts)
head(cpm(d))
apply(d$counts, 2, sum) # total gene counts per sample


# Method 1: use pyfaidx
d <- calcNormFactors(d)
# upgrade pip from 8.1.2 to 9.0.1
d
sudo pip install --upgrade pip
</pre>
sudo pip install pyfaidx
* [https://www.rdocumentation.org/packages/edgeR/versions/3.14.0/topics/calcNormFactors ?calcNormFactors]. If ‘object’ is a ‘matrix’, the output is a vector with length ‘ncol(object)’ giving the relative normalization factors.
$ cd /tmp
<pre>
$ faidx $GENOMEFA -i chromsizes > sizes.genome
y <- matrix( rpois(1000, lambda=5), nrow=200 )
$ cat sizes.genome
calcNormFactors(y)
Pt 154478
</pre>
Mt 366924
* [https://davetang.org/muse/2011/01/24/normalisation-methods-for-dge-data/ Normalisation methods implemented in edgeR]
4 18585056
* https://stats.stackexchange.com/a/421903, [https://bioconductor.org/packages/release/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf#page=27 Vignette]
2 19698289
<pre>
3 23459830
dge <- DGEList(M)
5 26975502
dge <- calcNormFactors(dge)
1 30427671
logCPM <- cpm(dge, log=TRUE)  # section 2.16 of vignette
# Method 2: use samtools
</pre>
$ /opt/SeqTools/bin/samtools-1.3/samtools faidx $GENOMEFA
 
$ cat $GENOMEFA.fai
== DESeq2 for DE analysis ==
Pt 154478 51 60 61
<ul>
Mt 366924 157155 60 61
<li>By default, DESeq(test = "Wald"). Another test option is LRT. </li>
4 18585056 530246 60 61
<li>[http://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html Vignette]. Keyword: Wald test, coefficients <math>\beta</math>, nbinomWaldTest. The coefficients βi give the log2 fold changes for gene i for each column of the model matrix X.
2 19698289 19425104 60 61
<pre>
3 23459830 39451749 60 61
# Suppose the condition has 2 levels
5 26975502 63302628 60 61
dds <- DESeqDataSetFromMatrix(countData = round(counts),
1 30427671 90727773 60 61
                              colData = DataFrame(condition),
$ cut -f1,2 $GENOMEFA.fai > sizes.genome
                              design = ~ condition)
$ cat sizes.genome2
dds <- DESeq(dds)
Pt 154478
res <- results(dds)
Mt 366924
res$stat  # same as coef(dds)[, 2]/coef(dds, SE=T)[, 2]
4 18585056
 
2 19698289
# coef(): extracting the matrix beta_{ir} for all genes i and model coefficients r
3 23459830
# In the case of 2 conditions, the 1st column of coef() is intercept
5 26975502
#    and the 2nd column of coef() is the condition.
1 30427671
all(res$stat == coef(dds)[, 2]/coef(dds, SE=T)[, 2], na.rm=T )
</syntaxhighlight>
# [1] TRUE
 
all(res$log2FoldChange == coef(dds)[, 2] )
all(res$lfcSE == coef(dds, SE=T)[, 2] )
</pre>
</li>
<li>'''log2FoldChange''' represents the log normalized counts from the mean of level 2 vs level 1. But it is not clear how to calculate that exactly. See [https://support.bioconductor.org/p/88813/ DESEq2 log2FoldChange and lfcSE calculations].
<li>According to '''?results''', The '''lfcSE''' gives the standard error of the '''log2FoldChange'''. For the '''Wald test''', '''stat''' is the Wald statistic: the log2FoldChange divided by lfcSE, which is compared to a standard Normal distribution to generate a two-tailed pvalue. </li>
<li>[https://hbctraining.github.io/DGE_workshop_salmon/lessons/05_DGE_DESeq2_analysis2.html Differential expression analysis with DESeq2] </li>
<li>Videos from Chipster Tutorials https://www.youtube.com/watch?v=5tGCBW3_0IA
 
<syntaxhighlight lang='rsplus'>
> samplesDESeq = with(samples, data.frame(
        shortname = I(shortname),
        countf = I(countf),
        condition = condition,
        LibraryLayout = LibraryLayout))


=== Remove/Add 'chr' from fasta ===
> samplesDESeq
<syntaxhighlight lang='bash'>
sed 's/chr//g' ucsc.hg19.fa >ucsc.hg19.nochr.fa
# or
cat hg19.fa | sed 's/>chr/>/g' > hg19_new.fa
</syntaxhighlight>


Similarly to add 'chr' to fasta:
shortname              countf condition LibraryLayout
<syntaxhighlight lang='bash'>
1  CT.PA.1  Untreated-3.count      CTL        PAIRED
cat hg19.fa | sed 's/>/>chr/g' > hg19_new.fa
2  CT.PA.2  Untreated-4.count      CTL        PAIRED
</syntaxhighlight>
3  KD.PA.3 CG8144_RNAi-3.count        KD        PAIRED
 
4  KD.PA.4 CG8144_RNAi-4.count        KD        PAIRED
=== Convert Fasta to Fastq ===
5  CT.SI.5  Untreated-1.count      CTL        SINGLE
It is OK to use Fasta file as the input sequencing files in alignment programs.
6  KD.SI.6 CG8144_RNAi-1.count        KD        SINGLE
7  CT.SI.7  Untreated-6.count      CTL        SINGLE
 
> library("DESeq")
cds <- newCountDataSetFromHTSeqCount(samplesDESeq) # quick, 15682 features, 7 samples
cds <- estimateSizeFactors(cds) # quick
sizeFactors(cds)


If we still want to convert FASTA to FASTQ, two methods are given with an example [[Genome#Convert_FASTA_to_FASTQ|here]]. Note quality scores are padded.
#   CT.PA.1  CT.PA.2  KD.PA.3  KD.PA.4  CT.SI.5  KD.SI.6  CT.SI.7
# 0.6991612 0.8104921 0.8217403 0.8941097 1.6431467 1.3720872 1.1041186


=== Convert Fastq to Fasta ===
cds <- estimateDispersions(cds) # quick
Note the quality scores are discarded.
res <- nbinomTest(cds,"CTL","KD") # 44 seconds
sum(res$pval <= .05, na.rm=T) # [1] 1574
sum(res$padj <= .05, na.rm=T) # [1] 730


* https://wikis.utexas.edu/display/bioiteam/Mapping+tutorial
options(width=100)
* https://www.biostars.org/p/85929/. Use '''cat test.fastq | paste - - - - | sed 's/^@/>/g'| cut -f1-2 | tr '\t' '\n' '''.
res[1:3,]
<syntaxhighlight lang='bash'>
#            id  baseMean baseMeanA baseMeanB foldChange log2FoldChange      pval padj
$ cat test.fastq
# 1 FBgn0000003  0.000000  0.000000  0.000000        NaN            NaN        NA  NA
@SRR002051.3554692 :8:290:671:993 length=33
# 2 FBgn0000008 88.526695 88.486694 88.580029  1.001055    0.001520941 0.9298627    1
CCATTCCCCAAAACAGTAAAACCACACACCGAG
# 3 FBgn0000014  3.054418  2.327533  4.023599  1.728697    0.789684778 0.6839858    1
+SRR002051.3554692 :8:290:671:993 length=33
</syntaxhighlight>
40&&+(&#$&3""%$#"#%$)%"%"%$/%%""#
</li>
@SRR002051.3554693 :8:290:941:550 length=33
</ul>
ACTAACATCCCATAATCGTACCTCCTACCATCA
 
+SRR002051.3554693 :8:290:941:550 length=33
=== DESeqDataSet methods ===
IIII*I$+(/II'$&:I"%""""&"&$%"'""I
<ul>
$ cat test.fastq | paste - - - - | sed 's/^@/>/g'| cut -f1-2 | tr '\t' '\n'
<li>counts(): This method returns the raw count data for the genes in the DESeqDataSet object.
>SRR002051.3554692 :8:290:671:993 length=33
<li>colData(): This method returns the metadata (e.g., sample information) for the samples in the DESeqDataSet object.
CCATTCCCCAAAACAGTAAAACCACACACCGAG
<li>rowRanges()
>SRR002051.3554693 :8:290:941:550 length=33
<li>design()
ACTAACATCCCATAATCGTACCTCCTACCATCA
<li>sizeFactors(): This method returns the size factors for the DESeqDataSet object, which are used to adjust for library size differences between samples.
<li>rld(): This method returns the regularized-logarithm (rlog) transformed data for the DESeqDataSet object, which is a variance-stabilizing transformation that can be used for visualization and clustering.
<li>plotDispEsts(): This method generates a plot of the dispersion estimates for the DESeqDataSet object, which can be used to diagnose the fit of the model.
<li>plotCounts(): This method generates a plot of the raw count data for the DESeqDataSet object, which can be used to visualize the distribution of the data.
<li>results(): This method returns the results of the differential expression analysis for the DESeqDataSet object, including the log2FoldChange values and the p-values.
<li>resultsNames()
<li>coef()
<li>plotMA(): MA plot
<li>Volcano plot:
<ul>
<li>[https://bioconductor.org/packages/release/bioc/vignettes/EnhancedVolcano/inst/doc/EnhancedVolcano.html EnhancedVolcano::EnhancedVolcano()]. Note:
# There are no default values for x and y parameters. All examples in the vignette use y = ‘pvalue’ (not ‘padj’).
# pCutoff means a p-value, not fdr adjustment
 
<syntaxhighlight lang='r'>
EnhancedVolcano(res,
    lab = rownames(res),
    pCutoff = 1e-05,
    FCcutoff = 1.0,
    x = 'log2FoldChange',
    y = 'pvalue')
</syntaxhighlight>
To draw adjusted p-values on the y-axis and use adjusted p-values as a cutoff (however, the label for some sig genes are missing. IOW, ggplot2 solution is better)
<syntaxhighlight lang='r'>
EnhancedVolcano(res,
    lab = rownames(res),
    pCutoff = 0.05,
    pCutoffCol = "padj",
    FCcutoff = 1.0,
    ylim = c(0, max(-log10(res$padj), na.rm = TRUE)),
    labSize = 2.5,
    x = 'log2FoldChange',
    y = 'padj',
    title = 'Volcano plot',
    ylab = bquote(~-Log[10]~'Adjusted p-value'),
    legendLabels = c('NS', 'Log2 FC', 'Adjusted p-value', 'Adjusted p-value & Log2 FC'))
</syntaxhighlight>
 
<li>ggplot2
<syntaxhighlight lang='r'>
ggplot(res, aes(x = log2FoldChange, y = -log10(pvalue))) +
  geom_point() +
  xlab("log2 Fold Change") + ylab("-log10(p-value)") +
ggtitle("Volcano Plot")
</syntaxhighlight>
If we like to add vertical and horizontal cutoff lines and change to use adjusted p-values ('''padj''' column), we can follow [https://groverj3.github.io/articles/2024-04-21_making-volcano-plots-with-ggplot2.html Making Volcano Plots With ggplot2]:
<syntaxhighlight lang='r'>
library(ggplot2)
library(ggrepel)
 
data <- res
data$gene <- rownames(res)
ggplot(data, aes(x = log2FoldChange, y = -log10(padj))) +
    geom_point(aes(color = padj < 0.05 & abs(log2FoldChange) > 1), alpha = 0.5) +
    scale_color_manual(values = c("black", "red"), na.translate = F) +
    geom_vline(
      xintercept = c(-1, 1),
      linetype = 'dashed'
    ) +
    geom_hline(
      yintercept = -log10(.05),
      linetype = 'dashed'
    ) +
    theme_bw() +
    labs(title = "Volcano Plot", x = "Log2 Fold Change", y = "-Log10 Adjusted P-Value") +
    geom_label_repel(
        data = significant_genes,
        aes(label = gene),
        size=3,
        box.padding = 0.25,    # default
        point.padding = 1e-06,  # default
        max.overlaps = 10      # default
    )
</syntaxhighlight>
</syntaxhighlight>
* [http://seqanswers.com/forums/showthread.php?t=5465 Convert fastq files to two fasta files with one containing the reads and another containing the quality scores]. In the example below we try to separate paired end reads generated by [[Genome#BEERS.2FGrant_G.R._2011|BEERS]] program.
</ul>
<syntaxhighlight lang='bash'>
</ul>
$ head simulated_reads_testbeers.fa
 
>seq.1a
=== Pre-filter ===
CGAAGAAGGACCCAAAGATGACAAGGCTCACAAAGTACACCCAGGGCAGTTCATACCCCATGGCATCTTGCATCCAGTAGAGCACATCGGTCCAGCCTTC
* [https://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#pre-filtering DESeq2 vignette]. row sum >= 10.  
>seq.1b
* [https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-022-05023-z Modeling and cleaning RNA-seq data significantly improve detection of differentially expressed genes] 2022.
GCTCGAGCTGTTCCTTGGACGAATGCACAAGACGTGCTACTTCCTGGGATCCGACATGGAAGCGGAGGAGGACCCATCGCCCTGTGCATCTTCGGGATCA
 
>seq.2a
=== betaPrior is FALSE by default ===
GCCCCAGCAGAGCCGGGTAAAGATCAGGAGGGTTAGAAAAAATCAGCGCTTCCTCTTCCTCCAAGGCAGCCAGACTCTTTAACAGGTCCGGAGGAAGCAG
[https://support.bioconductor.org/p/118257/ Different results in DESeq2 when using betaPrior=TRUE and betaPrior=FALSE]
>seq.2b
 
ATGAAGCCTTTTCCCATGGAGCCATATAACCATAATCCCTCAGAAGTCAAGGTCCCAGAATTCTACTGGGATTCTTCCTACAGCATGGCTGATAACAGAT
=== lfcShrink ===
>seq.3a
* [https://rdrr.io/bioc/DESeq2/man/lfcShrink.html ?lfcShrink]
CCCCAGAGGAGCGCCACCTGTCCAAGATGCAGCAGAACGGCTACGAAAATCCAACCTACAAGTTCTTTGAGCAGATGCAGAACTAGACCCCCGCCACAGC
* [https://support.bioconductor.org/p/9153664/ lfcShrink() and batch effects in DESeq2]
$ awk 'NR % 4 == 1 {print ">" $0 } NR % 4 == 2 {print $0}' simulated_reads_testbeers.fa > my.fasta
 
$ head my.fasta
=== Getting NAs in p-values ===
>>seq.1a
[https://support.bioconductor.org/p/9153862/ Getting NAs in both p-values (normal and adjusted) in DEseq2]
CGAAGAAGGACCCAAAGATGACAAGGCTCACAAAGTACACCCAGGGCAGTTCATACCCCATGGCATCTTGCATCCAGTAGAGCACATCGGTCCAGCCTTC
 
>>seq.2a
=== Comparison ===
GCCCCAGCAGAGCCGGGTAAAGATCAGGAGGGTTAGAAAAAATCAGCGCTTCCTCTTCCTCCAAGGCAGCCAGACTCTTTAACAGGTCCGGAGGAAGCAG
[https://www.tandfonline.com/doi/full/10.1080/02664763.2020.1788518 A comparison study on modeling of clustered and overdispersed count data for multiple comparisons] Kruppa 2020
>>seq.3a
 
CCCCAGAGGAGCGCCACCTGTCCAAGATGCAGCAGAACGGCTACGAAAATCCAACCTACAAGTTCTTTGAGCAGATGCAGAACTAGACCCCCGCCACAGC
=== Wald vs Likelihood ratio test ===
>>seq.4a
* [https://support.bioconductor.org/p/9159380/#9159382 HELP - DESeq2 difference between LRT and Wald Test in RNA-seq/ATAC-seq].
CGGGTGGAGGCTGGCCAAGCAACGAAGGAGATAAAATTGCAGCTGGCATTGTGTGTGCGCGCGCCCGCGGCGGCGGCCAGCAGGGGTGCGCGCGGAGCGG
** The Wald tests are specific to a given contrast. You are specifically asking if there is evidence that the coefficient comparing two groups is larger than zero... I would imagine that the vast majority of people use Wald tests exclusively, as they are mostly wanting to make specific comparisons.
>>seq.5a
** If you fit a full and reduced model and then compute a likelihood ratio test, you are testing if the full model fits the data better than the reduced model. It's an omnibus test that tells you that at least one coefficient is significant, much like an F-test in a conventional linear regression.
CGCTCACCTTCAATGTCACTGGCAAACATACTGCCTACCAGCTACTCCTCAGCCTTACACCTTAAGCATACGTCCTGCTCTTACTCTTCGAGTTGAGGGT
$ awk 'NR % 4 == 0 {print ">" $0 } NR % 4 == 3 {print $0}' simulated_reads_testbeers.fa > my.fasta
$ head my.fasta
>seq.1b
>GCTCGAGCTGTTCCTTGGACGAATGCACAAGACGTGCTACTTCCTGGGATCCGACATGGAAGCGGAGGAGGACCCATCGCCCTGTGCATCTTCGGGATCA
>seq.2b
>ATGAAGCCTTTTCCCATGGAGCCATATAACCATAATCCCTCAGAAGTCAAGGTCCCAGAATTCTACTGGGATTCTTCCTACAGCATGGCTGATAACAGAT
>seq.3b
>GTTTGATACAGCTAAATTCTTTACAGTACACAAAACCCATTAATAATGTAGTATAGAGACCAAAATGTAAAGAGAGATAGAATACATTACTGATGTGTGG
>seq.4b
>CGGGGCCCCGCCCGCGGCTAGGGAGGTGGCCGCCCTGGCCCGAGCCTGCTGCCCACGGCCGCGGCACTCAGCTCGCACTCCTTCAGCCCGGGAGGCCGGG
>seq.5b
>ACTCCCTGATAGTCTATGGAAGGAAAATGACAACTATTTTAGAATATTTCTAGTTTGTTTTTTCAGTGATCTTTTCATCCAGGCCTTGTTACTGTTACAG
</syntaxhighlight>
* https://gif.biotech.iastate.edu/converting-fastq-fasta
* Bioconductor packages '''Biostrings''' or '''shortread''' https://support.bioconductor.org/p/38338/


=== GATK ===
=== DESeq2, edgeR, limma ===
When I use Ensembl/GRCh37 as the reference to run GATK, I got an error
* [https://zhuanlan.zhihu.com/p/518142469 DESeq2, edgeR, limma 差异分析结果比较]
<pre style="white-space: pre-wrap; /* CSS 3 */ white-space: -moz-pre-wrap; /* Mozilla, since 1999 */ white-space: -pre-wrap; /* Opera 4-6 */ white-space: -o-pre-wrap; /* Opera 7 */ word-wrap: break-word; /* IE 5.5+ */ " >
* [https://www.biostars.org/p/284775/ How do I explain the difference between edgeR, LIMMA, DESeq etc. to experimental Biologist/non-bioinformatician].
##### ERROR MESSAGE: Input files /home/brb/SeqTestdata/usefulvcf/hg19/gatk/common_all_20160601.vcf and reference have incompatible contigs. Please see http://gatkforums.broadinstitute.org/discussion/63/input-files-have-incompatible-contigsfor more information. Error details: No overlapping contigs found.
** DESeq and EdgeR are very similar and both assume that no genes are differentially expressed.
##### ERROR  /home/brb/SeqTestdata/usefulvcf/hg19/gatk/common_all_20160601.vcf contigs = [chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22, chrX, chrY]
** Limma / Voom is different in that it normalises via the very successful (for microarrays) quantile nomalisation, where an attempt is made to match gene count distributions across samples in your dataset.
##### ERROR  reference contigs = [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, Y, MT]
* [https://ucdavis-bioinformatics-training.github.io/2018-June-RNA-Seq-Workshop/thursday/DE.html Differential Expression with Limma-Voom]
</pre>
 
The conclusion is I need to check if the chromosome name in dbsnp vcf file and reference fa file has the same naming convention.
=== DESeq2 paired multifactor test ===
[https://support.bioconductor.org/p/62357/ DESeq2 paired multifactor test]
 
=== t-test vs DESeq2 ===
[https://support.bioconductor.org/p/9150836/ FPKM t-test vs DEseq2]. t-tests are parametric, based on the normal distribution. t-tests are expected to yield far fewer DEGs simply because at low sample size they're massively underpowered, please read papers to learn why. That is the basis why methods such as DESeq2 even exist. RNA-seq is not normally distributed, that is well-known.
 
=== Time series ===
* [https://master.bioconductor.org/packages/release/workflows/vignettes/rnaseqGene/inst/doc/rnaseqGene.html#time-course-experiments RNA-seq workflow: gene-level exploratory analysis and differential expression]
* [http://onetipperday.sterding.com/2021/04/note-for-deseq2-time-course-analysis.html Note for DEseq2 time course analysis], [http://onetipperday.sterding.com/2021/07/note-2-for-deseq2-time-series-data.html Note 2]


For example, for Ensemble/GRCh37, I should NOT use the GATK version (i.e. no 'chr' was added) from ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh37p13/VCF/
=== Interaction ===
[https://support.bioconductor.org/p/9153193/ Complex DESeq2 Experimental Design]


(P.S. For UCSC hg19, we shall use the GATK version ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh37p13/VCF/GATK/)
= File Format =
* http://genome.ucsc.edu/FAQ/FAQformat.html which includes a lot of common formats like BAM, BED, bedGraph, bigBed, bigWig, VCF, WIG, et al.
* http://stephenturner.us/biofiletypes/  


But for UCSC/hg38, I should use the <span style="color: red">GATK version ('chr' was added)</span> from ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh38p2/VCF/GATK/. Read the [ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh38p2/VCF/GATK/GATK_VCF.readme readme]
== [http://en.wikipedia.org/wiki/FASTQ_format fastq] ==


So I end up with the file structure in my PC
The [http://en.wikipedia.org/wiki/FASTQ_format#Format_converters wikipedia] website provides information to convert FASTQ to FASTA format (when do we want to do that?).
* usefulvcf/hg19/common_all*.vcf  (no 'chr' was added)
* <strike>usefulvcf/hg38/gatk/common_all*.vcf</strike>  usefulvcf/hg38/chr/common_all*.vcf  ('chr' was added)


== [http://www.ensembl.org/info/website/upload/gff.html gff/gtf] ==
=== fastq format ===
* The GTF file can be downloaded from the Tophat website. After that, extract the file '''genes.gtf''' according to the instruction in [http://linus.nci.nih.gov/bdge/#preparingfiles BDGE] website.
A FASTQ file normally uses four lines per sequence.
* GTF file can also be downloaded from
** [http://hgdownload.soe.ucsc.edu/downloads.html UCSC Genome Bioinformatics] or [http://genome.ucsc.edu/cgi-bin/hgTables UCSC Table Browser]. See the Mapping reads to the genome tutorial in [http://homer.salk.edu/homer/basicTutorial/mapping.html homer.salk.edu].
** [ftp://ftp.ncbi.nih.gov/genomes/ NCBI]
** [http://useast.ensembl.org/info/data/ftp/index.html Ensembl]
* GTF file can change. For example there are 7 versions of hg19 reference genome,
<syntaxhighlight lang='bash'>
brb@T3600 ~ $ ls -l ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/
total 4
drwxrwxr-x 9 brb brb 4096 Jul 24  2015 Archives
lrwxrwxrwx 1 brb brb  30 Mar 10 03:03 Genes -> Archives/archive-current/Genes
lrwxrwxrwx 1 brb brb  35 Mar 10 03:03 README.txt -> Archives/archive-current/README.txt
lrwxrwxrwx 1 brb brb  33 Mar 10 03:03 SmallRNA -> Archives/archive-current/SmallRNA
lrwxrwxrwx 1 brb brb  34 Mar 10 03:03 Variation -> Archives/archive-current/Variation
brb@T3600 ~ $ ls -lH ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Archives
total 28
drwxrwxr-x 4 brb brb 4096 Mar 15  2012 archive-2010-09-27-22-25-17
drwxrwxr-x 5 brb brb 4096 Mar 15  2012 archive-2011-01-27-18-25-49
drwxrwxr-x 5 brb brb 4096 Mar 15  2012 archive-2011-08-30-21-45-18
drwxrwxr-x 5 brb brb 4096 Mar 15  2012 archive-2012-03-09-03-24-41
drwxrwxr-x 5 brb brb 4096 Oct  1  2013 archive-2013-03-06-11-23-03
drwxrwxr-x 5 brb brb 4096 Jun  2  2014 archive-2014-06-02-13-47-56
drwxrwxr-x 5 brb brb 4096 Jul 24  2015 archive-2015-07-17-14-32-32
lrwxrwxrwx 1 brb brb  27 Mar 10 03:35 archive-current -> archive-2015-07-17-14-32-32
</syntaxhighlight>
* GTF file is used in RNA-Seq reads alignment and count reads.
** [https://ccb.jhu.edu/software/tophat/manual.shtml Tophat] - If this option is provided, TopHat will first extract the '''transcript sequences''' and use Bowtie to align reads to this virtual transcriptome first. Only the reads that do not fully map to the '''transcriptome''' will then be mapped on the genome. The reads that did map on the transcriptome will be converted to genomic mappings (spliced as needed) and merged with the novel mappings and junctions in the final tophat output.
** [https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf STAR] - GTF file (containing '''annotated transcripts''') can be used in creating STAR index files ('''--sjdbGTFfile''' option in '''STAR --runMode genomeGenerate'''). STAR will extract '''splice junctions''' from this file and use them to greatly improve accuracy of the mapping. Note that in the mapping step there is no need to supply the GTF file again.
** [http://www-huber.embl.de/users/anders/HTSeq/doc/count.html htseq-count]: one of two required inputs (the other one is a sorted bam file).
* The meaning of Gene isoform in [http://en.wikipedia.org/wiki/Gene_isoform wikipedia]. It contains links to TSS, CDS, UTR.
* The meaning of transcriptome in [http://en.wikipedia.org/wiki/Transcriptome wikipedia]. Each row in a GTF file contains one feature and the the feature's transcript id and gene name. See [https://gist.github.com/arraytools/a3d2fdcd0e2ff37110a0 gist].
* Gene name in GTF file
<pre>
<pre>
$ grep KRAS ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf | wc -l
@SEQ_ID
23
GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT
+
!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65
</pre>
</pre>
* What is CDS in GTF file? '''CDS (contiguous sequence) CDS is the sequence that actually makes proteins. ''' See [https://www.biostars.org/p/65162/ biostars.org].
* Line 1 begins with a '@' character and is followed by a sequence identifier and an optional description (like a FASTA title line).
* What is exon? '''A region of the transcript sequence within a gene which is not removed from the primary RNA transcript by RNA splicing.''' See its definition on  
* Line 2 is the raw sequence letters.
** [http://www.sequenceontology.org/browser/current_svn/term/SO:0000147 sequenceontology.org]
* Line 3 begins with a '+' character and is optionally followed by the same sequence identifier (and any description) again.
** '''[http://ghr.nlm.nih.gov/glossary=exon U.S. National Library of Medicine]'''
* Line 4 encodes the quality values for the sequence in Line 2, and must contain the same number of symbols as letters in the sequence.
** [http://dictionary.reference.com/browse/exon?s=t dictionary]
 
**  [http://www.biology-online.org/dictionary/Exon biology-online.org]
It seems line 3 & 4 are not required. See the simulated fastq files on http://genomespot.blogspot.com/2014/11/dna-aligner-accuracy-bwa-bowtie-soap.html. I can use BWA to run alignment without a problem:)
** [http://www.news-medical.net/health/What-are-introns-and-exons.aspx new-medical.net].
 
* GFF3 format from [http://www.sequenceontology.org/resources/gff3.html sequenceontology.org]
=== Paired end ===
[http://www.cureffi.org/2012/12/19/forward-and-reverse-reads-in-paired-end-sequencing/ Forward and reverse reads in paired-end sequencing]


The following output shows how many features from different sources.
The paired files can be two formats. See [https://bioinf.comav.upv.es/courses/sequence_analysis/sequence_file_formats.html. Bioinformatics at COMAV]
<syntaxhighlight lang='bash'>
brb@brb-T3500:/tmp$ wc -l ~/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa
61913917 /home/brb/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa
brb@brb-T3500:/tmp$ wc -l ~/igenomes/Homo_sapiens/NCBI/build37.2/Sequence/WholeGenomeFasta/genome.fa
44224234 /home/brb/igenomes/Homo_sapiens/NCBI/build37.2/Sequence/WholeGenomeFasta/genome.fa
brb@brb-T3500:/tmp$ wc -l ~/igenomes/Homo_sapiens/Ensembl/GRCh37/Sequence/WholeGenomeFasta/genome.fa
51594937 /home/brb/igenomes/Homo_sapiens/Ensembl/GRCh37/Sequence/WholeGenomeFasta/genome.fa


brb@brb-T3500:~/igenomes/Homo_sapiens$ wc -l UCSC/hg19/Annotation/Genes/genes.gtf
Format1:
869204 UCSC/hg19/Annotation/Genes/genes.gtf
<pre>
brb@brb-T3500:~/igenomes/Homo_sapiens$ wc -l NCBI/build37.2/Annotation/Genes/genes.gtf
Fastq file 1
819119 NCBI/build37.2/Annotation/Genes/genes.gtf
@molecule_1 1st_read_from_pair
brb@brb-T3500:~/igenomes/Homo_sapiens$ wc -l Ensembl/GRCh37/Annotation/Genes/genes.gtf
@molecule_2 1st_read_from_pair
2280612 Ensembl/GRCh37/Annotation/Genes/genes.gtf
@molecule_3 1st_read_from_pair
</syntaxhighlight>
<syntaxhighlight lang='rsplus'>
brb@brb-T3500:~/igenomes/Homo_sapiens$ R
> x <- read.delim("~/igenomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf", header=FALSE, as.is=TRUE)
> dim(x)
[1] 2280612      9
> x[1:2, 1:8]
  V1                  V2        V3    V4    V5 V6 V7 V8
1  1 processed_transcript      exon 11869 12227  .  +  .
2  1 processed_transcript transcript 11869 14409  .  +  .
> strsplit(x[1:2, 9], ";") # not the same number of elements so we cannot apply as.data.frame()
[[1]]
[1] "exon_id ENSE00002234944"        " exon_number 1"
[3] " gene_biotype pseudogene"      " gene_id ENSG00000223972"
[5] " gene_name DDX11L1"            " gene_source ensembl_havana"
[7] " transcript_id ENST00000456328" " transcript_name DDX11L1-002"
[9] " transcript_source havana"      " tss_id TSS15145"


[[2]]
Fastq file 2
[1] "gene_biotype pseudogene"        " gene_id ENSG00000223972"
@molecule_1 2nd_read_from_pair
[3] " gene_name DDX11L1"            " gene_source ensembl_havana"
@molecule_2 2nd_read_from_pair
[5] " transcript_id ENST00000456328" " transcript_name DDX11L1-002"
@molecule_3 2nd_read_from_pair
[7] " transcript_source havana"      " tss_id TSS15145"
</pre>
 
Format 2:
> table(x[, 3])
<pre>
Interleaved Fastq file
@molecule_1 1st_read_from_pair
@molecule_1 2nd_read_from_pair
@molecule_2 1st_read_from_pair
@molecule_2 2nd_read_from_pair
@molecule_3 1st_read_from_pair
@molecule_3 2nd_read_from_pair
</pre>
 
Note from my experiment, if we flip the two fastq files order in the BWA command, the resulting bam file will diff at FLAG (2nd) column. The final vcf file is not changed.


        CDS        exon start_codon  stop_codon
=== FastQValidator ===
    794920    1309155      92839      83698
http://genome.sph.umich.edu/wiki/FastQValidator
> y <- read.delim("~/igenomes/Homo_sapiens/NCBI/build37.2/Annotation/Genes/genes.gtf", header=FALSE, as.is=TRUE)
> table(y[, 3])


        CDS        exon start_codon  stop_codon
== [http://en.wikipedia.org/wiki/FASTA_format fasta] ==
    345395      405671      34041      34012
> z <- read.delim("~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf", header=FALSE, as.is=TRUE)
> table(z[, 3])


        CDS        exon start_codon stop_codon
FASTA format is a text-based format for representing nucleotide sequences . cf. FASTQ files provide more information than FASTA since FASTQ format stores both a biological sequence (usually nucleotide sequence) and its corresponding quality scores.
    365947      430178      36547      36532


> options(width=120)
The reference genome is saved in FASTA format.
> table(x[, 2])


          3prime_overlapping_ncrna                          antisense                          IG_C_gene
Normally the first line in a FASTA file starts with a ">" (greater-than) symbol. Subsequent lines starting with a semicolon would be ignored by software. Following the initial line (used for a unique description of the sequence) is the actual sequence itself in standard one-letter code. A multiple sequence FASTA format would be obtained by concatenating several single sequence FASTA files.
                                92                              40326                                290
                  IG_C_pseudogene                          IG_D_gene                          IG_J_gene
                                40                                196                                78
                  IG_J_pseudogene                          IG_V_gene                    IG_V_pseudogene
                                12                              1099                                657
                          lincRNA                              miRNA                          misc_RNA
                            48588                              6848                              4380
                          Mt_rRNA                            Mt_tRNA            nonsense_mediated_decay
                                4                                44                            293471
                    non_stop_decay            polymorphic_pseudogene              processed_pseudogene
                              1132                              1853                              24516
              processed_transcript                    protein_coding                        pseudogene
                            172582                            1978244                              2247
                  retained_intron                              rRNA                    sense_intronic
                            150034                              1140                              2474
                sense_overlapping                            snoRNA                              snRNA
                              1255                              3242                              4148
  transcribed_processed_pseudogene transcribed_unprocessed_pseudogene    translated_processed_pseudogene
                              1182                              7931                                  3
                        TR_C_gene                          TR_D_gene                          TR_J_gene
                                58                                  9                                246
                  TR_J_pseudogene                          TR_V_gene                    TR_V_pseudogene
                                8                                869                                107
                unitary_pseudogene            unprocessed_pseudogene
                              1467                              13763
> table(y[, 2])


unknown
For the fruit fly genome file ''Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa''. The '''grep -n''' command can be used to get the row numbers of some keyword (chromosome names in this case).
819119
<pre>
> table(z[, 2])
$ head Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa
>2L dna:chromosome chromosome:BDGP5:2L:1:23011544:1 REF
CGACAATGCACGACAGAGGAAGCAGAACAGATATTTAGATTGCCTCTCATTTTCTCTCCC
ATATTATAGGGAGAAATATGATCGCGTATGCGAGAGTAGTGCCAACATATTGTGCTCTTT
GATTTTTTGGCAACCCAAAATGGTGGCGGATGAACGAGATGATAATATATTCAAGTTGCC
GCTAATCAGAAATAAATTCATTGCAACGTTAAATACAGCACAATATATGATCGCGTATGC
GAGAGTAGTGCCAACATATTGTGCTAATGAGTGCCTCTCGTTCTCTGTCTTATATTACCG
CAAACCCAAAAAGACAATACACGACAGAGAGAGAGAGCAGCGGAGATATTTAGATTGCCT
ATTAAATATGATCGCGTATGCGAGAGTAGTGCCAACATATTGTGCTCTCTATATAATGAC
TGCCTCTCATTCTGTCTTATTTTACCGCAAACCCAAATCGACAATGCACGACAGAGGAAG
CAGAACAGATATTTAGATTGCCTCTCATTTTCTCTCCCATATTATAGGGAGAAATATGAT


unknown
$ grep -n ">" Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa
869204
1:>2L dna:chromosome chromosome:BDGP5:2L:1:23011544:1 REF
</syntaxhighlight>
383528:>2LHet dna:chromosome chromosome:BDGP5:2LHet:1:368872:1 REF
389677:>2R dna:chromosome chromosome:BDGP5:2R:1:21146708:1 REF
742124:>2RHet dna:chromosome chromosome:BDGP5:2RHet:1:3288761:1 REF
796938:>3L dna:chromosome chromosome:BDGP5:3L:1:24543557:1 REF
1205999:>3LHet dna:chromosome chromosome:BDGP5:3LHet:1:2555491:1 REF
1248592:>3R dna:chromosome chromosome:BDGP5:3R:1:27905053:1 REF
1713678:>3RHet dna:chromosome chromosome:BDGP5:3RHet:1:2517507:1 REF
1755638:>4 dna:chromosome chromosome:BDGP5:4:1:1351857:1 REF
1778170:>U dna:chromosome chromosome:BDGP5:U:1:10049037:1 REF
1945655:>Uextra dna:chromosome chromosome:BDGP5:Uextra:1:29004656:1 REF
2429067:>X dna:chromosome chromosome:BDGP5:X:1:22422827:1 REF
2802782:>XHet dna:chromosome chromosome:BDGP5:XHet:1:204112:1 REF
2806185:>YHet dna:chromosome chromosome:BDGP5:YHet:1:347038:1 REF
2811970:>dmel_mitochondrion_genome dna:chromosome chromosome:BDGP5:dmel_mitochondrion_genome:1:19517:1 REF


An example from UCSC/hg19. As you can see from IGV and the output of the first few rows,
$ wc -l *.fa
* the locations of each rows are not exclusive. For example, rows #3 and #4 are overlapped that matches with what we saw on IGV (fly example)
2812296 Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa
* one row represents one feature; one transcript consists several features
* one gene (e.g. WASH7P) can be split over multiple regions (UCSC/hg19 example).
<pre>
$ more ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf
chr1    unknown exon    11874  12227  .      +      .      gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS16932";
chr1    unknown exon    12613  12721  .      +      .      gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS16932";
chr1    unknown exon    13221  14409  .      +      .      gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS16932";
chr1    unknown exon    14362  14829  .      -       .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    14970  15038  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    15796  15947  .      -      .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    16607  16765  .      -      .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    16858  17055  .       -      .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    17233  17368  .       -      .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    17369  17436  .      -      .      gene_id "MIR6859-3"; gene_name "MIR6859-3"; transcript_id "NR_107063_2"; tss_id "TSS25677";
chr1    unknown exon    17369  17436  .      -      .      gene_id "MIR6859-2"; gene_name "MIR6859-2"; transcript_id "NR_107062_1"; tss_id "TSS25677";
chr1    unknown exon    17369  17436  .      -      .      gene_id "MIR6859-4"; gene_name "MIR6859-4"; transcript_id "NR_128720_2"; tss_id "TSS25677";
chr1    unknown exon    17369  17436  .      -      .      gene_id "MIR6859-1"; gene_name "MIR6859-1"; transcript_id "NR_106918_2"; tss_id "TSS25677";
chr1    unknown exon    17606  17742  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    17915  18061  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    18268  18366  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    24738  24891  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    29321  29370  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    30366  30503  .      +      .      gene_id "MIR1302-11"; gene_name "MIR1302-11"; transcript_id "NR_036268"; tss_id "TSS10004";
</pre>
</pre>
[[File:Igv hg19.png|300px]]


The following table gives an example (UCSC/hg19) from one feature. '''Notice that the last column contains transcript ID, tss ID and gene ID. If the GTF is obtained from Ensembl, this column also has exon ID.'''
For human,
{| class="wikitable"
<pre>
| 1
$ grep -n ">" ~/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa
| seqname
1:>chrM
| chr1 (called 1 from NCBI & Ensembl)
334:>chr1
|-
4985348:>chr2
| 2
9849337:>chr3
| source
13809787:>chr4
| unknown
17632874:>chr5
|-
21251181:>chr6
| 3
24673484:>chr7
| feature
27856259:>chr8
| exon/transcript
30783541:>chr9
|-
33607811:>chr10
| 4
36318507:>chr11
| start
39018639:>chr12
| 11874
41695678:>chr13
|-
43999077:>chr14
| 5
46146069:>chr15
| end
48196698:>chr16
| 12227
50003795:>chr17
|-
51627701:>chr18
| 6
53189247:>chr19
| score
54371828:>chr20
| .
55632340:>chr21
|-
56594939:>chr22
| 7
57621032:>chrX
| strand
60726445:>chrY
| +
</pre>
|-
| 8
| frame
| 0
|-
| 9
| attribute
| gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS14844";
|}


The gene attributes (gene_id, transcript_id, exon_number, gene_name, gene_biotype, transcript_name, protein_id) appears on the mouse-over pop-up in IGV. See the 1st screenshot below from fly genome (imported manually by myself to IGV). From the 1st screenshot it is clear that one position may contain more than one transcripts.
=== chromosome names ===
 
When I apply the above method to examine the genome.fa files from Ensembl, NCBI and UCSC using the command
[[File:IGV fly Anders2013.png|300px]] [[File:IGV fly Anders2013 2.png|300px]]
<syntaxhighlight lang='bash'>
grep -n ">" genome.fa | cut -d' ' -f1 | cut -d '>' -f2
# for example
grep -n ">" ~/Downloads/Homo_sapiens.GRCh38.dna.chromosome.22.fa | cut -d' ' -f1 | cut -d '>' -f2 
# 22
</syntaxhighlight>
I get the following result
# Ensembl:
## GRCh37: 1, 2, 3, ...., X, Y, MT
## GRCh38: 1, 2, 3, ... (see note below)
# NCBI:
## build37.1, build37.2: 1, 2, 3, ....,X, Y, MT
## GRCh38: chr1, chr2, ...., chr22, chrX, chrY, chrM, chr1_KI270706v1_random, ....., chrEBV
# UCSC:
## hg18, hg19: chr1, chr2, ...., chr22, chrX, chrY, chrM
## hg38: chr1, chr2, ..., chr22, chrX, chrY, chrM, ..., chrUn_GL000218v1, chrEBV


[http://seqanswers.com/forums/showthread.php?t=29552 This post] on seqanswers.com has a discussion about the GTF file, exon, intron, CDS, exon, etc. It says
Note that
* <span style="color: red">Neither Illumina or Biowulf provide GRCh38 from Ensembl</span> (maybe for a good reason). Ensembl (http://ftp.ensembl.org/pub/release-76/fasta/homo_sapiens/dna) does provide GRCh38.
* GRCh38 from Ensembl is still following its GRCh37 convention; it won't include 'chr' on their chromosome names. That is, even the same name GRCh38 was used in both Ensembl and NCBI, they have different conventions.


'''"Exon" refers to transcription (DNA->RNA) and "CDS" to translation (RNA->Proteins). These are two different biological mechanisms.'''
=== chromosome sizes ===
https://www.biostars.org/p/134807/


=== Splice junction ===
<syntaxhighlight lang='bash'>
GTF file contains splice junction information. See [[Genome#Splice_junction|Genome > Splice junction]] on how to extract this information.
GENOMEFA=Arabidopsis_thaliana.TAIR10.23.dna.genome.fa


== bed ==
# Method 1: use pyfaidx
A BED file (.bed) is a tab-delimited text file that defines a feature track. Bed files can be like GTF/GFF to provide gene annotations. See [http://useast.ensembl.org/info/website/upload/bed.html?redirect=no ensembl.org]. Tracks in the UCSC Genome Browser (http://genome.ucsc.edu/) can be downloaded to BED files and loaded into IGV.
# upgrade pip from 8.1.2 to 9.0.1
 
sudo pip install --upgrade pip
A simple bed file looks like
sudo pip install pyfaidx
<pre>
$ cd /tmp
chr7    127471196  127472363
$ faidx $GENOMEFA -i chromsizes > sizes.genome
</pre>
$ cat sizes.genome
 
Pt 154478
We can also take a look some bed files generated after running the Tophat program.
Mt 366924
<pre>
4 18585056
brb@brb-T3500:~/Anders2013/Untreated-6$ head -n 3 insertions.bed
2 19698289
track name=insertions description="TopHat insertions"
3 23459830
2L      10457  10457  T      1
5 26975502
2L      10524  10524  T      1
1 30427671
brb@brb-T3500:~/Anders2013/Untreated-6$ head -n 3 deletions.bed
# Method 2: use samtools
track name=deletions description="TopHat deletions"
$ /opt/SeqTools/bin/samtools-1.3/samtools faidx $GENOMEFA
2L      10832  10833  -      1
$ cat $GENOMEFA.fai
2L      12432  12434  -       1
Pt 154478 51 60 61
brb@brb-T3500:~/Anders2013/Untreated-6$ head -n 3 junctions.bed
Mt 366924 157155 60 61
track name=junctions description="TopHat junctions"
4 18585056 530246 60 61
2L      11095  11479  JUNC00000001    4       -      11095  11479  255,0,0 2       74                                          ,70    0,314
2 19698289 19425104 60 61
2L      11271  11479  JUNC00000002    24      -      11271  11479  255,0,0 2      73                                          ,70    0,138
3 23459830 39451749 60 61
</pre>
5 26975502 63302628 60 61
1 30427671 90727773 60 61
$ cut -f1,2 $GENOMEFA.fai > sizes.genome
$ cat sizes.genome2
Pt 154478
Mt 366924
4 18585056
2 19698289
3 23459830
5 26975502
1 30427671
</syntaxhighlight>


From my observations, the IGV can create the junctions track (it is called 'Untreated-6_s.bam junctions' when I load the <Untreated-6_s.bam> file) automatically once we load the bam file. If we manually load the <junctions.bed> file saved in the 'Untreated-6' directory, we can see both junction tracks are the same.
R approach
<syntaxhighlight lang='rsplus'>
library(Biostrings)
x <- readDNAStringSet("~/igenomes/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa")
y <- width(x[1:24])
barplot(y, las = 2)
y
#    chr1      chr2      chr3      chr4      chr5      chr6      chr7      chr8
# 248956422 242193529 198295559 190214555 181538259 170805979 159345973 145138636
#      chr9    chr10    chr11    chr12    chr13    chr14    chr15    chr16
# 138394717 133797422 135086622 133275309 114364328 107043718 101991189  90338345
#    chr17    chr18    chr19    chr20    chr21    chr22      chrX      chrY
#  83257441  80373285  58617616  64444167  46709983  50818468 156040895  57227415
</syntaxhighlight>
[[File:Chromosome len.png|300px]]


== sam/bam, "samtools view" and Rsamtools ==
=== Remove/Add 'chr' from fasta ===
* http://biobits.org/samtools_primer.html
<syntaxhighlight lang='bash'>
* [[Genome#SAMtools|Genome-SAMtools]]
sed 's/chr//g' ucsc.hg19.fa >ucsc.hg19.nochr.fa
* http://samtools.github.io/hts-specs/SAMv1.pdf (especially p4)
# or
* http://genome.sph.umich.edu/wiki/SAM
cat hg19.fa | sed 's/>chr/>/g' > hg19_new.fa
* https://class.coursera.org/gencommand-003/lecture/39 (video)
</syntaxhighlight>
* See [[Genome#vcf_format|VCF format]]
 
* https://youtu.be/2KqBSbkfhRo?list=UUqaMSQd_h-2EDGsU6WDiX0Q (Bioconductor)
Similarly to add 'chr' to fasta:
<syntaxhighlight lang='bash'>
cat hg19.fa | sed 's/>/>chr/g' > hg19_new.fa
</syntaxhighlight>
 
=== Convert Fasta to Fastq ===
It is OK to use Fasta file as the input sequencing files in alignment programs.  
 
If we still want to convert FASTA to FASTQ, two methods are given with an example [[Genome#Convert_FASTA_to_FASTQ|here]]. Note quality scores are padded.


There are 11 required fields in the sam file and the header is optional. See also http://bio-bwa.sourceforge.net/bwa.shtml.
=== Convert Fastq to Fasta ===
Note the quality scores are discarded.


{| class="wikitable"
* https://wikis.utexas.edu/display/bioiteam/Mapping+tutorial
! column field
* https://www.biostars.org/p/85929/. Use '''cat test.fastq | paste - - - - | sed 's/^@/>/g'| cut -f1-2 | tr '\t' '\n' '''.
! example (/ExomeLungCancer/H1975cellLine)
<syntaxhighlight lang='bash'>
|-
$ cat test.fastq
| 1 QNAME
@SRR002051.3554692 :8:290:671:993 length=33
| SRR2923335.1
CCATTCCCCAAAACAGTAAAACCACACACCGAG
|-
+SRR002051.3554692 :8:290:671:993 length=33
| 2 FLAG
40&&+(&#$&3""%$#"#%$)%"%"%$/%%""#
| 99
@SRR002051.3554693 :8:290:941:550 length=33
|-
ACTAACATCCCATAATCGTACCTCCTACCATCA
| 3 RNAME (chromosome name)
+SRR002051.3554693 :8:290:941:550 length=33
| 6
IIII*I$+(/II'$&:I"%""""&"&$%"'""I
|-
$ cat test.fastq | paste - - - - | sed 's/^@/>/g'| cut -f1-2 | tr '\t' '\n'
| 4 POS
>SRR002051.3554692 :8:290:671:993 length=33
| 36938157
CCATTCCCCAAAACAGTAAAACCACACACCGAG
|-
>SRR002051.3554693 :8:290:941:550 length=33
| 5 MAPQ
ACTAACATCCCATAATCGTACCTCCTACCATCA
| 60
</syntaxhighlight>
|-
* [http://seqanswers.com/forums/showthread.php?t=5465 Convert fastq files to two fasta files with one containing the reads and another containing the quality scores]. In the example below we try to separate paired end reads generated by [[Genome#BEERS.2FGrant_G.R._2011|BEERS]] program.
| 6 CIGAR
<syntaxhighlight lang='bash'>
| 100M
$ head simulated_reads_testbeers.fa
|-
>seq.1a
| 7 RNEXT
CGAAGAAGGACCCAAAGATGACAAGGCTCACAAAGTACACCCAGGGCAGTTCATACCCCATGGCATCTTGCATCCAGTAGAGCACATCGGTCCAGCCTTC
| =
>seq.1b
|-
GCTCGAGCTGTTCCTTGGACGAATGCACAAGACGTGCTACTTCCTGGGATCCGACATGGAAGCGGAGGAGGACCCATCGCCCTGTGCATCTTCGGGATCA
| 8 PNEXT
>seq.2a
| 36938259
GCCCCAGCAGAGCCGGGTAAAGATCAGGAGGGTTAGAAAAAATCAGCGCTTCCTCTTCCTCCAAGGCAGCCAGACTCTTTAACAGGTCCGGAGGAAGCAG
|-
>seq.2b
| 9 TLEN
ATGAAGCCTTTTCCCATGGAGCCATATAACCATAATCCCTCAGAAGTCAAGGTCCCAGAATTCTACTGGGATTCTTCCTACAGCATGGCTGATAACAGAT
| 202
>seq.3a
|-
CCCCAGAGGAGCGCCACCTGTCCAAGATGCAGCAGAACGGCTACGAAAATCCAACCTACAAGTTCTTTGAGCAGATGCAGAACTAGACCCCCGCCACAGC
| 10 SEQ
$ awk 'NR % 4 == 1 {print ">" $0 } NR % 4 == 2 {print $0}' simulated_reads_testbeers.fa > my.fasta
| NGCAAGTAGAGGGGCGCACACCTACCCGCAGTTGTTCACAGCCATGAGGTCGCCAACTAGCAGGAAGGGGTAGGTCAGCATGCTCACTGCAATCTGAAAC
$ head my.fasta
|-
>>seq.1a
| 11 QUAL
CGAAGAAGGACCCAAAGATGACAAGGCTCACAAAGTACACCCAGGGCAGTTCATACCCCATGGCATCTTGCATCCAGTAGAGCACATCGGTCCAGCCTTC
| #1=DDFDFHHHHHJIJJJJIJJJJJJJJIJJJJJIJIHHHHHFFFFFEECEDDDDDDDDDDDDDDDDDDD&gt;BDDACCDDDDDDDDDDDDDDCCDDEDDDC
>>seq.2a
|-
GCCCCAGCAGAGCCGGGTAAAGATCAGGAGGGTTAGAAAAAATCAGCGCTTCCTCTTCCTCCAAGGCAGCCAGACTCTTTAACAGGTCCGGAGGAAGCAG
| 12 Tags (optional)
>>seq.3a
| NM:i:1 MD:Z:0G99 AS:i:99 XS:i:61 MQ:i:60
CCCCAGAGGAGCGCCACCTGTCCAAGATGCAGCAGAACGGCTACGAAAATCCAACCTACAAGTTCTTTGAGCAGATGCAGAACTAGACCCCCGCCACAGC
|}
>>seq.4a
 
CGGGTGGAGGCTGGCCAAGCAACGAAGGAGATAAAATTGCAGCTGGCATTGTGTGTGCGCGCGCCCGCGGCGGCGGCCAGCAGGGGTGCGCGCGGAGCGG
We can use '''samtools view''' to view the bam file ('''samtools view''' will not include the header, '''samtools view -H''' will include only the header, '''samtools view -h''' will include the header)
>>seq.5a
<syntaxhighlight lang='bash'>
CGCTCACCTTCAATGTCACTGGCAAACATACTGCCTACCAGCTACTCCTCAGCCTTACACCTTAAGCATACGTCCTGCTCTTACTCTTCGAGTTGAGGGT
$ samtools view -h accepted_hits.bam | more
$ awk 'NR % 4 == 0 {print ">" $0 } NR % 4 == 3 {print $0}' simulated_reads_testbeers.fa > my.fasta
@HD VN:1.4
$ head my.fasta
@SQ SN:1 LN:249250621
>seq.1b
@SQ SN:2 LN:243199373
>GCTCGAGCTGTTCCTTGGACGAATGCACAAGACGTGCTACTTCCTGGGATCCGACATGGAAGCGGAGGAGGACCCATCGCCCTGTGCATCTTCGGGATCA
@SQ SN:3 LN:198022430
>seq.2b
@SQ SN:4 LN:191154276
>ATGAAGCCTTTTCCCATGGAGCCATATAACCATAATCCCTCAGAAGTCAAGGTCCCAGAATTCTACTGGGATTCTTCCTACAGCATGGCTGATAACAGAT
@SQ SN:5 LN:180915260
>seq.3b
@SQ SN:6 LN:171115067
>GTTTGATACAGCTAAATTCTTTACAGTACACAAAACCCATTAATAATGTAGTATAGAGACCAAAATGTAAAGAGAGATAGAATACATTACTGATGTGTGG
@SQ SN:7 LN:159138663
>seq.4b
@SQ SN:8 LN:146364022
>CGGGGCCCCGCCCGCGGCTAGGGAGGTGGCCGCCCTGGCCCGAGCCTGCTGCCCACGGCCGCGGCACTCAGCTCGCACTCCTTCAGCCCGGGAGGCCGGG
@SQ SN:9 LN:141213431
>seq.5b
@SQ SN:10 LN:135534747
>ACTCCCTGATAGTCTATGGAAGGAAAATGACAACTATTTTAGAATATTTCTAGTTTGTTTTTTCAGTGATCTTTTCATCCAGGCCTTGTTACTGTTACAG
@SQ SN:11 LN:135006516
@SQ SN:12 LN:133851895
@SQ SN:13 LN:115169878
@SQ SN:14 LN:107349540
@SQ SN:15 LN:102531392
@SQ SN:16 LN:90354753
@SQ SN:17 LN:81195210
@SQ SN:18 LN:78077248
@SQ SN:19 LN:59128983
@SQ SN:20 LN:63025520
@SQ SN:21 LN:48129895
@SQ SN:22 LN:51304566
@SQ SN:X LN:155270560
@SQ SN:Y LN:59373566
@SQ SN:MT LN:16569
@PG ID:STAR PN:STAR VN:STAR_2.5.0c CL:STAR  --runThreadN 11  --genomeDir STARindex  --readFilesIn test.SRR493369
_1.fastq   test.SRR493369_2.fastq      --outFileNamePrefix ./LFB_HOXA1KD_repA/  --outSAMtype BAM  Unsorted      --twop
assMode Basic
@CO user command line: STAR --genomeDir STARindex --twopassMode Basic --runThreadN 11 --outSAMtype BAM Unsorted --ou
tFileNamePrefix ./LFB_HOXA1KD_repA/ --readFilesIn test.SRR493369_1.fastq test.SRR493369_2.fastq
SRR493369.1 99 19 3977426 255 1S100M = 3977605 488 ACTGGATCTCCACAAGGTAGATGGGCTCCATGAGGCGTGG
CTGGGCGGTCAGCACACTGGCGTAGAGGCAGCGCCGTGCTGTGGGGATGATCTGGCCCCCT CCCFFFFFHHHHHJJJJGIJJHIIJIJIJJIIJIJJJHJJJJJIIJDGDCDEDDDC
DDCDDDB@BDDDDDDDBBB@99@BD>CCDD<@BCCCD:3@##### NH:i:1 HI:i:1 AS:i:199 nM:i:1
SRR493369.1 147 19 3977605 255 4M208N97M = 3977426 -488 CGCCCTCCTTGGTGGCCCACTGGAAGCCGGCC
ACCACACTGTCCTTGATCTCGTTGAGGTACTGCACACCCTTGGTGATGTCGGTGAGGATGTTGGGGCCG 5BDDBBDDDDDDBABDDDCDDDDDDDCDDDDDCDCCCADEFFFFFHHH
</syntaxhighlight>
To count the number of rows in the header, use the Linux comand '''grep''' (though samtools view provides the '''-H''' argument to keep only the header)
<syntaxhighlight lang='bash'>
$ samtools view -h SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | grep "^@" | wc -l
$ # same as samtools view -H SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | wc -l
28
$ samtools view -h SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | grep -v "^@" | wc -l
$ # same as samtools view SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | wc -l
9856
$ samtools view -h SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | wc -l
9884
</syntaxhighlight>
</syntaxhighlight>
* https://gif.biotech.iastate.edu/converting-fastq-fasta
* Bioconductor packages '''Biostrings''' or '''shortread''' https://support.bioconductor.org/p/38338/
=== GATK ===
When I use Ensembl/GRCh37 as the reference to run GATK, I got an error
<pre style="white-space: pre-wrap; /* CSS 3 */ white-space: -moz-pre-wrap; /* Mozilla, since 1999 */ white-space: -pre-wrap; /* Opera 4-6 */ white-space: -o-pre-wrap; /* Opera 7 */ word-wrap: break-word; /* IE 5.5+ */ " >
##### ERROR MESSAGE: Input files /home/brb/SeqTestdata/usefulvcf/hg19/gatk/common_all_20160601.vcf and reference have incompatible contigs. Please see http://gatkforums.broadinstitute.org/discussion/63/input-files-have-incompatible-contigsfor more information. Error details: No overlapping contigs found.
##### ERROR  /home/brb/SeqTestdata/usefulvcf/hg19/gatk/common_all_20160601.vcf contigs = [chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22, chrX, chrY]
##### ERROR  reference contigs = [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, Y, MT]
</pre>
The conclusion is I need to check if the chromosome name in dbsnp vcf file (ftp://ftp.ncbi.nih.gov/snp/organisms/) and reference fa file has the same naming convention.
For example, for Ensemble/GRCh37, I should NOT use the GATK version (i.e. no 'chr' was added) from ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh37p13/VCF/
(P.S. For UCSC hg19, we shall use the GATK version ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh37p13/VCF/GATK/)
But for UCSC/hg38, I should use the <span style="color: red">GATK version ('chr' was added)</span> from ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh38p2/VCF/GATK/. Read the [ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh38p2/VCF/GATK/GATK_VCF.readme readme]


We can also use Bioconductor's [http://bioconductor.org/packages/release/bioc/html/Rsamtools.html Rsamtools] package to manipulate sam/bam, fasta, bcf and tabix files.
So I end up with the file structure in my PC
<syntaxhighlight lang='rsplus'>
* usefulvcf/hg19/common_all*.vcf  (no 'chr' was added)
source("https://bioconductor.org/biocLite.R")
* <strike>usefulvcf/hg38/gatk/common_all*.vcf</strike> usefulvcf/hg38/chr/common_all*.vcf  ('chr' was added)
biocLite("Rsamtools")
library(Rsamtools)
bf1 = BamFileList(file = c("file1.bam", "file2.bam"))
seqinfo(bf1)


# Suppose we want to get the MAPQ value from reads
== [http://www.ensembl.org/info/website/upload/gff.html gff/gtf] ==
param<-ScanBamParam(what="mapq") # other choices, what = c("rname", "strand", "pos", "qwidth", "seq")
* [https://www.r-bloggers.com/comparing-ensembl-gtf-and-cdna-2/ Comparing Ensembl GTF and cDNA]/ensembl's Fasta and GTF annotation files do not match
                                # see scanBamwhat()
* The GTF file can be downloaded from the Tophat website. After that, extract the file '''genes.gtf''' according to the instruction in [http://linus.nci.nih.gov/bdge/#preparingfiles BDGE] website.
bam<-scanBam(file="filename.bam", param=param)
* GTF file can also be downloaded from
str(bam)
** [http://hgdownload.soe.ucsc.edu/downloads.html UCSC Genome Bioinformatics] or [http://genome.ucsc.edu/cgi-bin/hgTables UCSC Table Browser]. See the Mapping reads to the genome tutorial in [http://homer.salk.edu/homer/basicTutorial/mapping.html homer.salk.edu].
summary(bam)
** [ftp://ftp.ncbi.nih.gov/genomes/ NCBI]
# List of 1
** [http://useast.ensembl.org/info/data/ftp/index.html Ensembl]
#  $ :List of 1
* GTF file can change. For example there are 7 versions of hg19 reference genome,
#  ..$ mapq: int [1:195875920] 0 13 0 0 2 0 0 0 9 0 ...
<syntaxhighlight lang='bash'>
object.size(bam)/2^20
brb@T3600 ~ $ ls -l ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/
# 747.207664489746 bytes
total 4
summary(bam[[1]][[1]])
drwxrwxr-x 9 brb brb 4096 Jul 24  2015 Archives
#    Min. 1st Qu. Median    Mean 3rd Qu.   Max.   NA's
lrwxrwxrwx 1 brb brb   30 Mar 10 03:03 Genes -> Archives/archive-current/Genes
#    0.0    60.0    60.0    58.4    60.0    60.0 2434291
lrwxrwxrwx 1 brb brb  35 Mar 10 03:03 README.txt -> Archives/archive-current/README.txt
table(bam[[1]][[1]])
lrwxrwxrwx 1 brb brb  33 Mar 10 03:03 SmallRNA -> Archives/archive-current/SmallRNA
 
lrwxrwxrwx 1 brb brb  34 Mar 10 03:03 Variation -> Archives/archive-current/Variation
        0        1        2        3        4         5        6        7
brb@T3600 ~ $ ls -lH ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Archives
   3186501    59642    40093    91541    52501    43220    123651    81155
total 28
        8        9        10       11        12        13        14        15
drwxrwxr-x 4 brb brb 4096 Mar 15  2012 archive-2010-09-27-22-25-17
    23354    133441    19302    17924    61882    30326    17755    54093
drwxrwxr-x 5 brb brb 4096 Mar 15  2012 archive-2011-01-27-18-25-49
      16        17        18        19        20        21        22        23
drwxrwxr-x 5 brb brb 4096 Mar 15  2012 archive-2011-08-30-21-45-18
    24826    21290    52880    37874    15116    69845    25458    30183
drwxrwxr-x 5 brb brb 4096 Mar 15  2012 archive-2012-03-09-03-24-41
      24        25        26        27        28       29        30        31
drwxrwxr-x 5 brb brb 4096 Oct  1  2013 archive-2013-03-06-11-23-03
    57684    80430    13746    537584    18437    14125    41978    25748
drwxrwxr-x 5 brb brb 4096 Jun  2  2014 archive-2014-06-02-13-47-56
      32        33        34        35        36        37        38        39
drwxrwxr-x 5 brb brb 4096 Jul 24  2015 archive-2015-07-17-14-32-32
    11848    63802    13745    16987    33168    29840    12198    64799
lrwxrwxrwx 1 brb brb  27 Mar 10 03:35 archive-current -> archive-2015-07-17-14-32-32
      40        41       42        43        44        45        46        47
  763253    32690    61987    46020    64149    85350    129188    100040
      48        49        50        51        52        53        54        55
  193770    683051    64096    95453    58853    87942    58469    48584
      56        57        58        59        60
    50132    135261    70818    101295 185161256
</syntaxhighlight>
</syntaxhighlight>
 
* GTF file is used in RNA-Seq reads alignment and count reads.
=== Flag ===
** [https://ccb.jhu.edu/software/tophat/manual.shtml Tophat] - If this option is provided, TopHat will first extract the '''transcript sequences''' and use Bowtie to align reads to this virtual transcriptome first. Only the reads that do not fully map to the '''transcriptome''' will then be mapped on the genome. The reads that did map on the transcriptome will be converted to genomic mappings (spliced as needed) and merged with the novel mappings and junctions in the final tophat output.
https://broadinstitute.github.io/picard/explain-flags.html Interpret FLAG value (interactive)
** [https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf STAR] - GTF file (containing '''annotated transcripts''') can be used in creating STAR index files ('''--sjdbGTFfile''' option in '''STAR --runMode genomeGenerate'''). STAR will extract '''splice junctions''' from this file and use them to greatly improve accuracy of the mapping. Note that in the mapping step there is no need to supply the GTF file again.
 
** [http://www-huber.embl.de/users/anders/HTSeq/doc/count.html htseq-count]: one of two required inputs (the other one is a sorted bam file).
For single end data, no flag is set so the flag is always 0.
* The meaning of Gene isoform in [http://en.wikipedia.org/wiki/Gene_isoform wikipedia]. It contains links to TSS, CDS, UTR.
 
* The meaning of transcriptome in [http://en.wikipedia.org/wiki/Transcriptome wikipedia]. Each row in a GTF file contains one feature and the the feature's transcript id and gene name. See [https://gist.github.com/arraytools/a3d2fdcd0e2ff37110a0 gist].
=== Extract by read names with grep ===
* Gene name in GTF file
<pre>
$ grep KRAS ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf | wc -l
23
</pre>
* What is CDS in GTF file? '''CDS (contiguous sequence) CDS is the sequence that actually makes proteins. ''' See [https://www.biostars.org/p/65162/ biostars.org].
* What is exon? '''A region of the transcript sequence within a gene which is not removed from the primary RNA transcript by RNA splicing.''' See its definition on
** [http://www.sequenceontology.org/browser/current_svn/term/SO:0000147 sequenceontology.org]
** '''[http://ghr.nlm.nih.gov/glossary=exon U.S. National Library of Medicine]'''
** [http://dictionary.reference.com/browse/exon?s=t dictionary]
**  [http://www.biology-online.org/dictionary/Exon biology-online.org]
** [http://www.news-medical.net/health/What-are-introns-and-exons.aspx new-medical.net].
* GFF3 format from [http://www.sequenceontology.org/resources/gff3.html sequenceontology.org]
 
The following output shows how many features from different sources.
<syntaxhighlight lang='bash'>
<syntaxhighlight lang='bash'>
# Extract reads SRR2923335.1 and SRR2923335.1999 and output only the read name (1st) and mapping quality columns (5th)
brb@brb-T3500:/tmp$ wc -l ~/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa
# -e = --extended-regexp, -w = matching the whole word
61913917 /home/brb/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa
# awk print $1,$5 will output with a space character between columns
brb@brb-T3500:/tmp$ wc -l ~/igenomes/Homo_sapiens/NCBI/build37.2/Sequence/WholeGenomeFasta/genome.fa
samtools view accepted_hits.bam  | grep -E -w "SRR2923335.1|SRR2923335.1999" | awk '{print $1,$5}'
44224234 /home/brb/igenomes/Homo_sapiens/NCBI/build37.2/Sequence/WholeGenomeFasta/genome.fa
brb@brb-T3500:/tmp$ wc -l ~/igenomes/Homo_sapiens/Ensembl/GRCh37/Sequence/WholeGenomeFasta/genome.fa
51594937 /home/brb/igenomes/Homo_sapiens/Ensembl/GRCh37/Sequence/WholeGenomeFasta/genome.fa
 
brb@brb-T3500:~/igenomes/Homo_sapiens$ wc -l UCSC/hg19/Annotation/Genes/genes.gtf
869204 UCSC/hg19/Annotation/Genes/genes.gtf
brb@brb-T3500:~/igenomes/Homo_sapiens$ wc -l NCBI/build37.2/Annotation/Genes/genes.gtf
819119 NCBI/build37.2/Annotation/Genes/genes.gtf
brb@brb-T3500:~/igenomes/Homo_sapiens$ wc -l Ensembl/GRCh37/Annotation/Genes/genes.gtf
2280612 Ensembl/GRCh37/Annotation/Genes/genes.gtf
</syntaxhighlight>
</syntaxhighlight>


=== Extract by a region ===
An example from UCSC/hg19. As you can see from IGV and the output of the first few rows,
<syntaxhighlight lang='rsplus'>
* the locations of each rows are not exclusive. For example, rows #3 and #4 are overlapped that matches with what we saw on IGV (fly example)
# https://www.biostars.org/p/48719/
* one row represents one feature; one transcript consists several features
# the output will be all reads starting anywhere between 18000-45500.
* one gene (e.g. WASH7P) can be split over multiple regions (UCSC/hg19 example).
samtools view input.bam "Chr10:18000-45500" > output.bam
<pre>
$ more ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf
chr1    unknown exon    11874  12227  .      +      .      gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS16932";
chr1    unknown exon    12613  12721  .      +      .      gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS16932";
chr1    unknown exon    13221  14409  .      +      .      gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS16932";
chr1    unknown exon    14362  14829  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    14970  15038  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    15796  15947  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    16607  16765  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    16858  17055  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    17233  17368  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    17369  17436  .      -      .      gene_id "MIR6859-3"; gene_name "MIR6859-3"; transcript_id "NR_107063_2"; tss_id "TSS25677";
chr1    unknown exon    17369  17436  .      -      .      gene_id "MIR6859-2"; gene_name "MIR6859-2"; transcript_id "NR_107062_1"; tss_id "TSS25677";
chr1    unknown exon    17369  17436  .      -      .      gene_id "MIR6859-4"; gene_name "MIR6859-4"; transcript_id "NR_128720_2"; tss_id "TSS25677";
chr1    unknown exon    17369  17436  .      -      .      gene_id "MIR6859-1"; gene_name "MIR6859-1"; transcript_id "NR_106918_2"; tss_id "TSS25677";
chr1    unknown exon    17606  17742  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    17915  18061  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    18268  18366  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    24738  24891  .      -      .      gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    29321  29370  .      -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    30366  30503  .      +      .       gene_id "MIR1302-11"; gene_name "MIR1302-11"; transcript_id "NR_036268"; tss_id "TSS10004";
</pre>
[[File:Igv hg19.png|300px]]


# https://www.biostars.org/p/45936/
The following table gives an example (UCSC/hg19) from one feature. '''Notice that the last column contains transcript ID, tss ID and gene ID. If the GTF is obtained from Ensembl, this column also has exon ID.'''
# index first!!!
{| class="wikitable"
samtools view -h reads.bam 1:1042000-1042010 > subset.sam
| 1
samtools view -h -b reads.bam chr1:10420000-10421000 > subset.bam
| seqname
</syntaxhighlight>
| chr1 (called 1 from NCBI & Ensembl)
 
|-
=== chromosomes ===
| 2
We can use the shell command in [https://www.biostars.org/p/14044/ this post] to extract the chromosome name, start position from the bam file.  There we can see the difference of the chromosome name of bam files created from using Ensembl and UCSC.
| source
<syntaxhighlight lang='bash'>
| unknown
# Ensembl GRCh37
|-
brb@T3600 ~/SeqTestdata/RNASeqFibroblast $ samtools view LFB_scramble_repA/accepted_hits.bam | head | awk '{print $3}'
| 3
1
| feature
1
| exon/transcript
1
|-
1
| 4
1
| start
| 11874
|-
| 5
| end
| 12227
|-
| 6
| score
| .
|-
| 7
| strand
| +
|-
| 8
| frame
| 0
|-
| 9
| attribute
| gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS14844";
|}


# NCBI GRCh38
The gene attributes (gene_id, transcript_id, exon_number, gene_name, gene_biotype, transcript_name, protein_id) appears on the mouse-over pop-up in IGV. See the 1st screenshot below from fly genome (imported manually by myself to IGV). From the 1st screenshot it is clear that one position may contain more than one transcripts.
brb@T3600 ~/SeqTestdata/RNASeqFibroblast $ samtools view RNAseq_Bam_ncbigrch38/LFB_scramble_repA.bam | head | awk '{print $3}'
chr1
chr1
chr1
chr1
chr1
chr1


# UCSC hg38
[[File:IGV fly Anders2013.png|300px]] [[File:IGV fly Anders2013 2.png|300px]]
brb@T3600 ~/SeqTestdata/RNASeqFibroblast $ samtools view RNAseq_Bam_hg38/LFB_scramble_repA.bam | head | awk '{print $3}'
chr1
chr1
chr1
chr1
chr1
chr1
</syntaxhighlight>


=== Subset reads from specific chromosomes ===
[http://seqanswers.com/forums/showthread.php?t=29552 This post] on seqanswers.com has a discussion about the GTF file, exon, intron, CDS, exon, etc. It says
https://www.biostars.org/p/128967/


For example to remove mitochondrial reads from BAM files
'''"Exon" refers to transcription (DNA->RNA) and "CDS" to translation (RNA->Proteins). These are two different biological mechanisms.'''
<syntaxhighlight lang='bash'>
samtools view -b input.bam chr1 chr2 chr3 chr4 > output.bam
# Or if the bam file has been indexed (i.e. .bai file is available)
samtools idxstats input.bam | cut -f 1 | grep -v MT | xargs samtools view -b input.bam > output.bam
</syntaxhighlight>
where samtools idxstat returns 4 columns (TAB-delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads).
<syntaxhighlight lang='bash'>
$ samtools idxstats input.bam | head
1 249250621 0 0
2 243199373 0 0
3 198022430 0 0
4 191154276 0 0
5 180915260 0 0
6 171115067 0 0
7 159138663 0 0
8 146364022 0 0
9 141213431 0 0
10 135534747 0 0
</syntaxhighlight>


=== Count the number of reads with certain number of mismatches ===
=== rtracklayer package ===
* https://www.biostars.org/p/130256/ Bwa appears to include only mismatches, but bowtie includes insertions and deletions in its '''NM'''.
See also the vignette of [https://bioconductor.org/packages/release/workflows/html/SingscoreAMLMutations.html SingscoreAMLMutations]
* https://gist.github.com/davfre/8596159
<pre>
brb@brb-T3500:~/igenomes/Homo_sapiens$ R
> x <- read.delim("~/igenomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf", header=FALSE, as.is=TRUE)
> dim(x)
[1] 2280612      9
> x[1:2, 1:8]
  V1                  V2        V3    V4    V5 V6 V7 V8
1  1 processed_transcript      exon 11869 12227  .  +  .
2  1 processed_transcript transcript 11869 14409  .  +  .
> strsplit(x[1:2, 9], ";") # not the same number of elements so we cannot apply as.data.frame()
[[1]]
[1] "exon_id ENSE00002234944"        " exon_number 1"
[3] " gene_biotype pseudogene"      " gene_id ENSG00000223972"
[5] " gene_name DDX11L1"            " gene_source ensembl_havana"
[7] " transcript_id ENST00000456328" " transcript_name DDX11L1-002"
[9] " transcript_source havana"      " tss_id TSS15145"


= Other alignment methods =
[[2]]
== [https://github.com/alexdobin/STAR STAR] ==
[1] "gene_biotype pseudogene"        " gene_id ENSG00000223972"
* Current [https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf Manual] (pdf is embedded in github)
[3] " gene_name DDX11L1"            " gene_source ensembl_havana"
* [http://labshare.cshl.edu/shares/gingeraslab/www-data/dobin/STAR/STAR.posix/doc/STARmanual.pdf Version 2.4 Manual] (pdf is searchable)
[5] " transcript_id ENST00000456328" " transcript_name DDX11L1-002"
[7] " transcript_source havana"      " tss_id TSS15145"


== Subread ==
> table(x[, 3])


== [http://ccb.jhu.edu/software/hisat2/index.shtml Hisat] ==
        CDS        exon start_codon  stop_codon
    794920    1309155      92839      83698
> y <- read.delim("~/igenomes/Homo_sapiens/NCBI/build37.2/Annotation/Genes/genes.gtf", header=FALSE, as.is=TRUE)
> table(y[, 3])
 
        CDS        exon start_codon  stop_codon
    345395      405671      34041      34012
> z <- read.delim("~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf", header=FALSE, as.is=TRUE)
> table(z[, 3])
 
        CDS        exon start_codon  stop_codon
    365947      430178      36547      36532
 
> options(width=120)
> table(x[, 2])


== Command line usages ==
          3prime_overlapping_ncrna                          antisense                          IG_C_gene
{| class="wikitable"
                                92                              40326                                290
! Aligner
                  IG_C_pseudogene                          IG_D_gene                          IG_J_gene
! Command Line
                                40                                196                                78
|-
                  IG_J_pseudogene                          IG_V_gene                    IG_V_pseudogene
| bowtie2
                                12                              1099                                657
| bowtie2 -x /path/to/bowtie2/BASENAME -1 A_1.fq,B_1.fq -2 A_2.fq,B_2.fq -S alignment.sam
                          lincRNA                              miRNA                          misc_RNA
|-
                            48588                              6848                              4380
| tophat2
                          Mt_rRNA                            Mt_tRNA            nonsense_mediated_decay
| tophat2 --no-coverage-search -p $SLURM_CPUS_PER_TASK \
                                4                                44                            293471
    -o OutputDir \
                    non_stop_decay            polymorphic_pseudogene              processed_pseudogene
    /path/to/bowtie2/BASENAME \
                              1132                              1853                              24516
    R1.FASTQ.gz R2.FASTQ.gz
              processed_transcript                    protein_coding                        pseudogene
|-
                            172582                            1978244                              2247
| STAR
                  retained_intron                              rRNA                    sense_intronic
| mkdir STARindex
                            150034                              1140                              2474
STAR --runMode genomeGenerate \
                sense_overlapping                            snoRNA                              snRNA
  --runThreadsN $SLURM_CPUS_PER_TASK \
                              1255                              3242                              4148
  --genomeDir STARindex \
  transcribed_processed_pseudogene transcribed_unprocessed_pseudogene   translated_processed_pseudogene
  --genomeFastaFiles /path/to/fasta.fa \
                              1182                              7931                                  3
   --sjdbGTFfile /path/to/genes.gtf \
                        TR_C_gene                          TR_D_gene                          TR_J_gene
  --sdjbOverhang 85
                                58                                  9                                246
STAR --twopassMode Basic \
                  TR_J_pseudogene                          TR_V_gene                    TR_V_pseudogene
  --runThreadN $SLURM_CPUS_PER_TASK \
                                8                                869                                107
  --genomeDir STARindex \
                unitary_pseudogene            unprocessed_pseudogene
  --outSAMmapqUnique 50 \
                              1467                              13763
  --sjdbOverhang 85 \
> table(y[, 2])
  --readFilesIn r1.fastq.gz r2.fastq.gz \
  --readFilesCommand zcat \
  --outSAMtype BAM Unsorted \
  --outFileNamePrefix output/star
|-
| Subread
| subread-buildindex -o INDEX /path/to/fasta.fa
subread-align -T $SLURM_CPUS_PER_TASK -i INDEX -r R1.FASTQ.gz -R R2.FASTQ.gz -o output/alignment.sam
|-
| Hisat2
| hisat2-build -p $SLURM_CPUS_PER_TASK \
  /path/to/fasta.fa INDEX
hisat2 -p $SLURM_CPUS_PER_TASK -x INDEX \
  -1 R1.FASTQ.gz \
  -2 R2.FASTQ.gz -S output/alignment.sam
|}


= What's special with rna-seq count data =
unknown
== Overdispersion ==
819119
* http://genomicsclass.github.io/book/pages/rnaseq_gene_level.html
> table(z[, 2])
* [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081415 Dispersion Estimation and Its Effect on Test Performance in RNA-seq Data Analysis: A Simulation-Based Comparison of Methods] by Landau 2013.


== Normalization ==
unknown
* [http://www.hindawi.com/journals/bmri/2015/621690/ The Impact of Normalization Methods on RNA-Seq Data Analysis] by Zyprych-Walczak 2015. Two public datasets (Bodymap and Cheung) are available from [http://bowtie-bio.sourceforge.net/recount/ recount] website.
869204
* [http://www.biomedcentral.com/1471-2105/11/94/ Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments] by Bullard 2010.
</pre>
* [http://www.biomedcentral.com/1471-2105/16/347 Comparing the normalization methods for the differential analysis of Illumina high-throughput RNA-Seq data] by Peipei Li 2015.
* [http://www.rna-seqblog.com/comparison-of-tmm-edger-rle-deseq2-and-mrn-normalization-methods/ Comparison of TMM (edgeR), RLE (DESeq2), and MRN Normalization Methods] by Maza E 2016
* [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169594 A Hypothesis Testing Based Method for Normalization and Differential Expression Analysis of RNA-Seq Data] by Zhou 2017


== Power Calculation ==
Another example. [https://davetang.github.io/muse/read_gtf.html Read GTF file into R].
[http://journal.frontiersin.org/article/10.3389/fgene.2016.00225/full PROPER: Extending the R Library PROPER to Enable Power Calculations for Isoform-Level Analysis with EBSeq]
<syntaxhighlight lang='rsplus'>
library(rtracklayer)
x <- import("/fdb/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf")
x
# GRanges object with 1006819 ranges and 9 metadata columns:
length(unique(x$gene_name))
# [1] 26364
length(unique(x$transcript_id))
# [1] 54070


= Other RNA-Seq data =
y <- read.delim("/fdb/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf")
dim(y)
# [1] 1006818      9
colnames(y)
# [1] "chr1"
# [2] "unknown"
# [3] "exon"
# [4] "X11874"
# [5] "X12227"
# [6] "."
# [7] "X."
# [8] "..1"
# [9] "gene_id.DDX11L1..gene_name.DDX11L1..transcript_id.NR_046018..tss_id.TSS16932."
</syntaxhighlight>


== GEO ==
=== Convert transcript to symbol ===
Go to http://www.ncbi.nlm.nih.gov/sites/entrez and select 'GEO Datasets' from the drop down menu, and use 'RNA-Seq' as your search term. http://www.ncbi.nlm.nih.gov/gds?term=rna-seq
<ul>
<li>Check out XXX.genes.results or XXX.isoforms.results file from RSEM output
<pre>
$ head Sample1.genes.results
gene_id transcript_id(s) length effective_length expected_count TPM FPKM
A1BG NM_130786 1766.00 1589.99 0.00 0.00 0.00
A1BG-AS1 NR_015380 2130.00 1953.99 0.00 0.00 0.00


* https://www.biostars.org/p/46059/
$ head Sample1.isoforms.results
* https://www.biostars.org/p/52866/
transcript_id gene_id length effective_length expected_count TPM FPKM IsoPct
NM_130786 A1BG 1766 1589.99 0.00 0.00 0.00 0.00
NR_015380 A1BG-AS1 2130 1953.99 0.00 0.00 0.00 0.00
</pre>
</li>
<li>[https://biotools.fr/human/ucsc_id_converter biotools] (online) </li>
<li>[https://support.bioconductor.org/p/9139532/ Converting between UCSC id and gene symbol with bioconductor annotation resources]. You need to use the [https://bioconductor.org/packages/release/data/annotation/html/Homo.sapiens.html Homo.sapiens] package.
<pre>
library(Homo.sapiens)
select(Homo.sapiens, "uc001qvk.1", "SYMBOL","TXNAME")
# 'select()' returned 1:1 mapping between keys and columns
#      TXNAME SYMBOL
# 1 uc001qvk.1    A2M
</pre>
</li>
<li>[https://www.biostars.org/p/332849/ How to map UCSC transcripts to gene symbol?] [https://bioconductor.org/packages/release/data/annotation/html/org.Hs.eg.db.html org.Hs.eg.db] package.
</li>
</ul>
=== Splice junction ===
GTF file contains splice junction information. See [[Genome#Splice_junction|Genome > Splice junction]] on how to extract this information.


=== GSE28666 ===
== bed ==
as used in DAFS paper. The timing is
A BED file (.bed) is a tab-delimited text file that defines a feature track. Bed files can be like GTF/GFF to provide gene annotations. See [http://useast.ensembl.org/info/website/upload/bed.html?redirect=no ensembl.org].  Tracks in the UCSC Genome Browser (http://genome.ucsc.edu/) can be downloaded to BED files and loaded into IGV.
* bowtie2-build 115 minutes
* fastq-dump 25 minutes per fastq (3.3 - 5.9GB fastq files)
* tophat2 90 minutes per fastq
* samtools 5 minutes
* htseq-count 13 minutes


=== GSE30567 ===
A simple bed file looks like
Used in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530905/ STAR] paper
<pre>
chr7    127471196  127472363
</pre>


=== GSE38886 ===
The format is explained in [https://genome.ucsc.edu/FAQ/FAQformat.html#format1 UCSC].
Used in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530905/ STAR] paper


=== GSE48215 & GSE48216 ===
We can also take a look some bed files generated after running the Tophat program.
[https://genomebiology.biomedcentral.com/articles/10.1186/gb-2013-14-10-r110 Modeling precision treatment of breast cancer]
<pre>
brb@brb-T3500:~/Anders2013/Untreated-6$ head -n 3 insertions.bed
track name=insertions description="TopHat insertions"
2L      10457  10457  T      1
2L      10524  10524  T      1
brb@brb-T3500:~/Anders2013/Untreated-6$ head -n 3 deletions.bed
track name=deletions description="TopHat deletions"
2L      10832  10833  -      1
2L      12432  12434  -      1
brb@brb-T3500:~/Anders2013/Untreated-6$ head -n 3 junctions.bed
track name=junctions description="TopHat junctions"
2L      11095  11479  JUNC00000001    4      -       11095  11479  255,0,0 2      74                                          ,70    0,314
2L      11271  11479  JUNC00000002    24      -       11271  11479  255,0,0 2      73                                          ,70    0,138
</pre>


GSE48215 is Whole exome Seq and GSE48216 is RNA-Seq
From my observations, the IGV can create the junctions track (it is called 'Untreated-6_s.bam junctions' when I load the <Untreated-6_s.bam> file) automatically once we load the bam file. If we manually load the <junctions.bed> file saved in the 'Untreated-6' directory, we can see both junction tracks are the same.


=== GSE51403 (full R script) ===
== sam/bam, "samtools view" and Rsamtools ==
[http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51403 RNA-seq differential expression studies: more sequence, or more replication?]
* http://biobits.org/samtools_primer.html
* [[Genome#SAMtools|Genome-SAMtools]]
* http://samtools.github.io/hts-specs/SAMv1.pdf (especially p4)
* http://genome.sph.umich.edu/wiki/SAM
* https://class.coursera.org/gencommand-003/lecture/39 (video)
* See [[Genome#vcf_format|VCF format]]
* https://youtu.be/2KqBSbkfhRo?list=UUqaMSQd_h-2EDGsU6WDiX0Q (Bioconductor)


An example of R code to download SRA files.
There are 11 required fields in the sam file and the header is optional. See also http://bio-bwa.sourceforge.net/bwa.shtml.
<pre>
        homo sapiens,
        Illumina HiSeq 2000.
        Question: what reference genome I should use?
        Ans1: hg18 was used.
              See http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1244821
              Or the paper.


        Ans2: GRCh38:
{| class="wikitable"
              Click 'BioProject' in http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51403
! column field
                then click 'Genome' in http://www.ncbi.nlm.nih.gov/bioproject/PRJNA222975
! example (/ExomeLungCancer/H1975cellLine)
                But tophat website only provides GRCh37.
|-
                https://genome.ucsc.edu/goldenPath/newsarch.html shows GRCh38 assembly is released
| 1 QNAME
                in Dec/2013.
| SRR2923335.1
 
|-
        38 fastq/SRR  (all are single lane)
| 2 FLAG
        14 samples/GSM
| 99
 
|-
        GSM#    SRX#    Sample          SRR#
| 3 RNAME (chromosome name)
      ---------------------------------------------------------------
| 6
Ctrl    1244822 365217  Ctrl_Rep7      1012952, 1012953, 1012954 (3 runs, 70M spots, 3.5G bases)
|-
        1244821 365216  Ctrl_Rep6      949-951 (I just skip prefix '1012')
| 4 POS
        1244820 365215  Ctrl_Rep5      946-948 (3 runs)
| 36938157
        1244819 365214  Ctrl_Rep4      943-945 (3 runs)  Control ethanol 24h
|-
        1244818 365213  Ctrl_Rep3      940-942 (3 runs)
| 5 MAPQ
        1244817 365212  Ctrl_Rep2      937-939 (3 runs)
| 60
        1244816 365211  Ctrl_Rep1      934-936 (3 runs)  Control ethanol 24h
|-
 
| 6 CIGAR
Trmnt  1244815 365210  E2_Rep7        931-933 (3 runs)  10nM E2 treatment for 24h
| 100M
        1244814 365209  E2_Rep6        928-930 (3 runs)
|-
        1244813 365208  E2_Rep5        925-927 (3 runs)
| 7 RNEXT
        1244812 365207  E2_Rep4        923-924 (2 runs)
| =
        1244811 365206  E2_Rep3        921-922 (2 runs)
|-
        1244810 365205  E2_Rep2        919-920 (2 runs)  10nM E2 treatment for 24h
| 8 PNEXT
        1244809 365204  E2_Rep1        917-918 (2 runs) 10nM E2 treatment for 24h
| 36938259
</pre>
|-
<syntaxhighlight lang='rsplus'>
| 9 TLEN
# Step 1. Download and convert data
| 202
|-
| 10 SEQ
| NGCAAGTAGAGGGGCGCACACCTACCCGCAGTTGTTCACAGCCATG…
|-
| 11 QUAL
| #1=DDFDFHHHHHJIJJJJIJJJJJJJJIJJJJJIJIHHHHHFFF…
|-
| 12 Tags (optional)
| NM:i:1 MD:Z:0G99 AS:i:99 XS:i:61 MQ:i:60
|}


library(SRAdb)
We can use '''samtools view''' to view the bam file ('''samtools view''' will not include the header, '''samtools view -H''' will include only the header, '''samtools view -h''' will include the header)
setwd("~/GSE51403")
<syntaxhighlight lang='bash'>
if( ! file.exists('SRAmetadb.sqlite') ) {
$ samtools view -h accepted_hits.bam | more
        # sqlfile <- getSRAdbFile()
@HD VN:1.4
  sqlfile <- "~/Anders2013/SRAmetadb.sqlite"
@SQ SN:1 LN:249250621
} else {
@SQ SN:2 LN:243199373
sqlfile <- 'SRAmetadb.sqlite'
@SQ SN:3 LN:198022430
}
@SQ SN:4 LN:191154276
sra_con <- dbConnect(SQLite(),sqlfile)
@SQ SN:5 LN:180915260
fs <- listSRAfile("SRP031476", sra_con, fileType = "sra" )    # 38 files
@SQ SN:6 LN:171115067
# getSRAfile("SRP031476", sra_con, fileType = "sra" )          # starting to download
@SQ SN:7 LN:159138663
 
@SQ SN:8 LN:146364022
getSRAfile(fs$run[13], sra_con, fileType='sra') # SRR1012917.sra
@SQ SN:9 LN:141213431
getSRAfile(fs$run[30], sra_con, fileType='sra') # SRR1012918.sra
@SQ SN:10 LN:135534747
 
@SQ SN:11 LN:135006516
dbDisconnect( sra_con )
@SQ SN:12 LN:133851895
 
@SQ SN:13 LN:115169878
fs <- dir(pattern='sra')
@SQ SN:14 LN:107349540
# for(f in fs)  system(paste("fastq-dump --split-3", f)) # Single thread, Not efficient
@SQ SN:15 LN:102531392
scrp <- paste("/opt/RNA-Seq/bin/sratoolkit.2.3.5-2-ubuntu64/bin/fastq-dump --split-3", fs)
@SQ SN:16 LN:90354753
 
@SQ SN:17 LN:81195210
library(parallel)
@SQ SN:18 LN:78077248
cl <- makeCluster(getOption("cl.cores", 5))
@SQ SN:19 LN:59128983
# note that if something is wrong, we need to delete broken fastq first and then run again.
@SQ SN:20 LN:63025520
system.time(clusterApply(cl, scrp, function(x) system(x)))
@SQ SN:21 LN:48129895
stopCluster(cl)
@SQ SN:22 LN:51304566
@SQ SN:X LN:155270560
@SQ SN:Y LN:59373566
@SQ SN:MT LN:16569
@PG ID:STAR PN:STAR VN:STAR_2.5.0c CL:STAR  --runThreadN 11  --genomeDir STARindex  --readFilesIn test.SRR493369
_1.fastq  test.SRR493369_2.fastq      --outFileNamePrefix ./LFB_HOXA1KD_repA/   --outSAMtype BAM  Unsorted      --twop
assMode Basic
@CO user command line: STAR --genomeDir STARindex --twopassMode Basic --runThreadN 11 --outSAMtype BAM Unsorted --ou
tFileNamePrefix ./LFB_HOXA1KD_repA/ --readFilesIn test.SRR493369_1.fastq test.SRR493369_2.fastq
SRR493369.1 99 19 3977426 255 1S100M = 3977605 488 ACTGGATCTCCACAAGGTAGATGGGCTCCATGAGGCGTGG
CTGGGCGGTCAGCACACTGGCGTAGAGGCAGCGCCGTGCTGTGGGGATGATCTGGCCCCCT CCCFFFFFHHHHHJJJJGIJJHIIJIJIJJIIJIJJJHJJJJJIIJDGDCDEDDDC
DDCDDDB@BDDDDDDDBBB@99@BD>CCDD<@BCCCD:3@##### NH:i:1 HI:i:1 AS:i:199 nM:i:1
SRR493369.1 147 19 3977605 255 4M208N97M = 3977426 -488 CGCCCTCCTTGGTGGCCCACTGGAAGCCGGCC
ACCACACTGTCCTTGATCTCGTTGAGGTACTGCACACCCTTGGTGATGTCGGTGAGGATGTTGGGGCCG 5BDDBBDDDDDDBABDDDCDDDDDDDCDDDDDCDCCCADEFFFFFHHH
</syntaxhighlight>
To count the number of rows in the header, use the Linux comand '''grep''' (though samtools view provides the '''-H''' argument to keep only the header)
<syntaxhighlight lang='bash'>
$ samtools view -h SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | grep "^@" | wc -l
$ # same as samtools view -H SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | wc -l
28
$ samtools view -h SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | grep -v "^@" | wc -l
$ # same as samtools view SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | wc -l
9856
$ samtools view -h SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | wc -l
9884
</syntaxhighlight>


# Step 2. Create samples object
We can also use Bioconductor's [http://bioconductor.org/packages/release/bioc/html/Rsamtools.html Rsamtools] package to manipulate sam/bam, fasta, bcf and tabix files.
samples <- data.frame(LibraryName = c(paste("Ctrl_Rep", 7:1, sep=''),
<syntaxhighlight lang='rsplus'>
                                      paste("E2_Rep", 7:1, sep='')),
source("https://bioconductor.org/biocLite.R")
            LibraryLayout = rep("SINGLE", 14),
biocLite("Rsamtools")
            fastq1=c(paste(paste("SRR", 1012952:1012954, ".fastq", sep=''), collapse=','),
library(Rsamtools)
                      paste(paste("SRR", 1012949:1012951, ".fastq", sep=''), collapse=','),
bf1 = BamFileList(file = c("file1.bam", "file2.bam"))
                      paste(paste("SRR", 1012946:1012948, ".fastq", sep=''), collapse=','),
seqinfo(bf1)
                      paste(paste("SRR", 1012943:1012945, ".fastq", sep=''), collapse=','),
 
                      paste(paste("SRR", 1012940:1012942, ".fastq", sep=''), collapse=','),
# Suppose we want to get the MAPQ value from reads
                      paste(paste("SRR", 1012937:1012939, ".fastq", sep=''), collapse=','),
param<-ScanBamParam(what="mapq") # other choices, what = c("rname", "strand", "pos", "qwidth", "seq")
                      paste(paste("SRR", 1012934:1012936, ".fastq", sep=''), collapse=','),
                                # see scanBamwhat()  
                      paste(paste("SRR", 1012931:1012933, ".fastq", sep=''), collapse=','),
bam<-scanBam(file="filename.bam", param=param)
                      paste(paste("SRR", 1012928:1012930, ".fastq", sep=''), collapse=','),
str(bam)
                      paste(paste("SRR", 1012925:1012927, ".fastq", sep=''), collapse=','),
summary(bam)
                      paste(paste("SRR", 1012923:1012924, ".fastq", sep=''), collapse=','),
# List of 1
                      paste(paste("SRR", 1012921:1012922, ".fastq", sep=''), collapse=','),
#  $ :List of 1
                      paste(paste("SRR", 1012919:1012920, ".fastq", sep=''), collapse=','),
#   ..$ mapq: int [1:195875920] 0 13 0 0 2 0 0 0 9 0 ...
                      paste(paste("SRR", 1012917:1012918, ".fastq", sep=''), collapse=',')),
object.size(bam)/2^20
            fastq2=rep("", 14),
# 747.207664489746 bytes
            countf=c(paste("Ctrl_Rep", 7:1, ".count", sep=''),
summary(bam[[1]][[1]])
                      paste("E2_Rep", 7:1, ".count", sep='')))
#   Min. 1st Qu.  Median    Mean 3rd Qu.   Max.   NA's
# Step 3: run tophat2
#     0.0    60.0    60.0    58.4    60.0    60.0 2434291
i <- 10
table(bam[[1]][[1]])
system.time(paste("tophat2 -G genes.gtf -p 5 -o",
                  samples$LibraryName[i],
                  "genome",
                  samples$fastq1[i]))
# system("tophat2 -G genes.gtf -p 5 -o E2_Rep5 genome SRR1012925.fastq")
# system("tophat2 -G genes.gtf -p 5 -o E2_Rep5 genome SRR1012925.fastq,SRR1012926.fastq,SRR1012927.fastq")
for(i in c(1:14)) {
  system(paste("tophat2 -G genes.gtf -p 5 -o", samples$LibraryName[i], "genome", samples$fastq1[i]))
}


# Step 4. Organize, sort and index the BAM files and create SAM files.
        0        1        2        3        4         5        6        7
for(i in seq_len(nrow(samples)) {
  3186501    59642    40093    91541    52501    43220    123651    81155
  lib = samples$LibraryName[i]
        8        9        10        11        12        13        14        15
  ob = file.path(lib, "accepted_hits.bam")
    23354    133441    19302    17924    61882    30326    17755    54093
  # sort by name, convert to SAM for htseq-count
      16        17        18        19        20        21        22        23
  c1 <- paste0("samtools sort -n ", ob, " ", lib, "_sn")
    24826    21290    52880    37874    15116    69845    25458    30183
  system(c1)
      24        25        26        27        28        29        30        31
}
    57684    80430    13746    537584    18437    14125    41978    25748
      32        33        34        35        36        37        38        39
    11848    63802    13745    16987    33168    29840    12198    64799
      40        41        42        43        44        45        46        47
  763253    32690    61987    46020    64149    85350    129188    100040
      48        49        50        51        52        53        54        55
  193770    683051    64096    95453    58853    87942    58469    48584
      56        57        58        59        60
    50132    135261    70818    101295 185161256
</syntaxhighlight>


samtools sort -n Sample1/accepted_hits.bam Sample1_sn  # 283 seconds
=== Flag ===
# samtools sort Sample1/accepted_hits.bam Sample1_s
https://broadinstitute.github.io/picard/explain-flags.html Interpret FLAG value (interactive)


# Step 5. Count reads using htseq-count
For single end data, no flag is set so the flag is always 0.
htseq-count -f bam -s no -a 10 Sample1_sn.bam Mus_musculus.GRCm38.70.gtf > Sample1.count # 759 seconds
htseq-count -f bam -s no -a 10 Sample2_sn.bam Mus_musculus.GRCm38.70.gtf > Sample2.count
samples$countsf <- paste(samples$LibraryName, "count", sep=".")
gf <- "genes.gtf"
cmd <- paste0("htseq-count -s no -a 10 ", samples$LibraryName, "_sn.sam ", gf, " > ", samples$countf)
system.time(clusterApply(cl, cmd, function(x) system(x))) # 119 minutes


=== Extract by read names with grep ===
<syntaxhighlight lang='bash'>
# Extract reads SRR2923335.1 and SRR2923335.1999 and output only the read name (1st) and mapping quality columns (5th)
# -e = --extended-regexp, -w = matching the whole word
# awk print $1,$5 will output with a space character between columns
samtools view accepted_hits.bam  | grep -E -w "SRR2923335.1|SRR2923335.1999" | awk '{print $1,$5}'
</syntaxhighlight>


samplesDESeq = data.frame(shortname = c("Sample1", "Sample2"), countf=c("Sample1.count", "Sample2.count"),
=== Extract by a region ===
                        condition = c("CTL", "KD"), LibraryLayout =c("SINGLE", "SINGLE"))
<syntaxhighlight lang='rsplus'>
library("DESeq")
# https://www.biostars.org/p/48719/
cds = newCountDataSetFromHTSeqCount(samplesDESeq)
# the output will be all reads starting anywhere between 18000-45500.
cds = estimateSizeFactors(cds)
samtools view input.bam "Chr10:18000-45500" > output.bam
sizeFactors(cds)
 
# https://www.biostars.org/p/45936/
# index first!!!
samtools view -h reads.bam 1:1042000-1042010 > subset.sam
samtools view -h -b reads.bam chr1:10420000-10421000 > subset.bam
</syntaxhighlight>
</syntaxhighlight>


=== GSE37544 ===
=== chromosomes ===
[http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37544 RNA-seq analysis of differential gene expression in liver from lactating dairy cows divergent in negative energy balance]
We can use the shell command in [https://www.biostars.org/p/14044/ this post] to extract the chromosome name, start position from the bam file. There we can see the difference of the chromosome name of bam files created from using Ensembl and UCSC.
<syntaxhighlight lang='bash'>
# Ensembl GRCh37
brb@T3600 ~/SeqTestdata/RNASeqFibroblast $ samtools view LFB_scramble_repA/accepted_hits.bam | head | awk '{print $3}'
1
1
1
1
1


=== GSE11045 ===
# NCBI GRCh38
[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126545 Probe Region Expression Estimation for RNA-Seq Data for Improved Microarray Comparability]
brb@T3600 ~/SeqTestdata/RNASeqFibroblast $ samtools view RNAseq_Bam_ncbigrch38/LFB_scramble_repA.bam | head | awk '{print $3}'
 
chr1
=== [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37703 GSE37703/SRP012607] ===
chr1
Differential analysis of HOXA1 in adult cells at isoform resolution by RNA-Seq by Trapnell C, Hendrickson DG, Sauvageau M, Goff L et al. Nat Biotechnol 2013.
chr1
chr1
chr1
chr1


The <samples.txt> file required by BRB-SeqTools looks like
# UCSC hg38
<pre>
brb@T3600 ~/SeqTestdata/RNASeqFibroblast $ samtools view RNAseq_Bam_hg38/LFB_scramble_repA.bam | head | awk '{print $3}'
LibraryName LibraryLayout fastq1 fastq2 ReadGroup SequencerManufacturer
chr1
LFB_scramble_repA PAIRED SRR493366_1.fastq SRR493366_2.fastq 1 illumina
chr1
LFB_scramble_repB PAIRED SRR493367_1.fastq SRR493367_2.fastq 2 illumina
chr1
LFB_scramble_repC PAIRED SRR493368_1.fastq SRR493368_2.fastq 3 illumina
chr1
LFB_HOXA1KD_repA PAIRED SRR493369_1.fastq SRR493369_2.fastq 4 illumina
chr1
LFB_HOXA1KD_repB PAIRED SRR493370_1.fastq SRR493370_2.fastq 5 illumina
chr1
LFB_HOXA1KD_repC PAIRED SRR493371_1.fastq SRR493371_2.fastq 6 illumina
</syntaxhighlight>
</pre>


=== GSE37704 ===
=== Subset reads from specific chromosomes ===
http://www.gettinggeneticsdone.com/2015/12/tutorial-rna-seq-differential.html
https://www.biostars.org/p/128967/


=== GSE64570, GSE69244, GSE72165 ===
For example to remove mitochondrial reads from BAM files
[http://f1000research.com/articles/4-1521/v1#DS3 Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences]
<syntaxhighlight lang='bash'>
samtools view -b input.bam chr1 chr2 chr3 chr4 > output.bam
# Or if the bam file has been indexed (i.e. .bai file is available)
samtools idxstats input.bam | cut -f 1 | grep -v MT | xargs samtools view -b input.bam > output.bam
</syntaxhighlight>
where samtools idxstat returns 4 columns (TAB-delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads).
<syntaxhighlight lang='bash'>
$ samtools idxstats input.bam | head
1 249250621 0 0
2 243199373 0 0
3 198022430 0 0
4 191154276 0 0
5 180915260 0 0
6 171115067 0 0
7 159138663 0 0
8 146364022 0 0
9 141213431 0 0
10 135534747 0 0
</syntaxhighlight>


=== Ion Torrent ===
=== Count the number of reads with certain number of mismatches ===
* [http://www.ncbi.nlm.nih.gov/geo/browse/?view=platforms&display=500&search=ion%20torrent&zsort=date GEO]
* https://www.biostars.org/p/130256/ Bwa appears to include only mismatches, but bowtie includes insertions and deletions in its '''NM'''.
* [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE46876 GSE46876] from GPL17301 PGM (Homo sapiens) and GPL17303 Ion Torrent Proton (Homo sapiens)
* https://gist.github.com/davfre/8596159
* [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE83668 GSE83668] from GPL17303 Ion Torrent Proton (Homo sapiens) and GPL11154 Illumina HiSeq 2000 (Homo sapiens)


== SRA ==
= Other alignment methods =
== Online tools (global and local alignments) ==
https://www.ebi.ac.uk/Tools/psa/


=== SRA000299 ===
== [https://github.com/alexdobin/STAR STAR] ==
* [http://genome.cshlp.org/content/18/9/1509.full Paper]
* Current [https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf Manual] (pdf is embedded in github)
* [http://davetang.org/muse/2012/05/09/getting-started-with-tophat/ Blog]  
* [http://labshare.cshl.edu/shares/gingeraslab/www-data/dobin/STAR/STAR.posix/doc/STARmanual.pdf Version 2.4 Manual] (pdf is searchable)


Note that one fastq file can represent several '''channels/lanes'''. The lane information can be extracted from fastq definition line (first line of each read). For example,
== Kallisto ==
<pre>
* https://pachterlab.github.io/kallisto/about
@SRR002321.1 080226_CMLIVERKIDNEY_0007:2:1:115:885
* Used in [http://www.biorxiv.org/content/biorxiv/early/2017/09/14/189092.full.pdf ARCHS4]
</pre>
"SRR002321.1" is the short read archive name, where the .1 is the read number, "080226_CMLIVERKIDNEY_0007" should be the Machine name, which has been arbitrarily changed
* "2" is the Channel/lane number
* "1" is the Tile number
* "115" is the X position
* "885" is the Y position
The website also shows how to extract reads with the same '''run''' from fastq files.


In this example, there are
== Subread ==
* two full sequencing runs (this info can be obtained from paper)
See [[Genome#Subread_.28RNA.2FDNA.29|Subread]].
* 4 experiments (SRX000571, SRX000604, SRX000605, SRX000606)
* 6 runs (SRRR002321, SRR002323, SRR002322, SRR002320, SRR002325, SRR002324)
* 7 lanes (eg "080226_CMLIVERKIDNEY_0007:2") for each full sequencing run


=== SRA data linked from [http://athen.cecad.uni-koeln.de/quickngs/web/download/ QuickNGS] ===
== [http://ccb.jhu.edu/software/hisat2/index.shtml Hisat] ==
Include Chip-Seq, RNA-Seq, WGS and miRNA-Seq. QuickNGS is a production environment for quick batch-wise analysis of Next-Generation Sequencing (NGS) data in high-throughput laboratories.


== [http://www.ebi.ac.uk/ena European Nucleotide Archive/ENA] ==
== Command line usages ==
 
{| class="wikitable"
== [http://www.ddbj.nig.ac.jp/ DDBJ] ==
! Aligner
 
! Command Line
== ENCODE ==
|-
http://www.ncbi.nlm.nih.gov/geo/info/ENCODE.html has a list of data with GSE number.
| bowtie2
 
| bowtie2 -x /path/to/bowtie2/BASENAME -1 A_1.fq,B_1.fq -2 A_2.fq,B_2.fq -S alignment.sam
[https://www.encodeproject.org/pipelines/ Data Processing Pipelines]
|-
 
| tophat2
== [http://www.1000genomes.org 1000 genomes] ==
| tophat2 --no-coverage-search -p $SLURM_CPUS_PER_TASK \
 
    -o OutputDir \
== Human BodyMap 2.0 data from Illumina ==
    /path/to/bowtie2/BASENAME \
* http://www.ensembl.info/blog/2011/05/24/human-bodymap-2-0-data-from-illumina/
    R1.FASTQ.gz R2.FASTQ.gz
* https://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise. Galaxy needs to install hg19 genome before we can use tophat or bowtie. See [https://wiki.galaxyproject.org/Admin/DataPreparation?action=show&redirect=Admin%2FNGS+Local+Setup Galaxy/Wiki/Admin/Data Preparation] page. Genome can be downloaded from [http://tophat.cbcb.umd.edu/igenomes.shtml tophat] webpage. It is 20GB for hg19! We can also download just chromosome 19 fasta file [http://hgdownload.cse.ucsc.edu/goldenPath/hg19/chromosomes/ chr19.fa.gz] from [http://hgdownload.cse.ucsc.edu/downloads.html UCSC genomes FTP server] and it is only 19MB. We can then create reference index files (.bt2) by using bowtie2-build command. If we use Galaxy, we don't need to create reference index files.
|-
* [http://www.biomedcentral.com/1471-2164/16/97 Impact of gene annotation on RNA-Seq data analysis]
| STAR
 
| mkdir STARindex
== [https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp TCGA/The Cancer Genome Atlas] ==
STAR --runMode genomeGenerate \
* [http://gdac.broadinstitute.org/ Broad Institute TCGA GDAC]
  --runThreadsN $SLURM_CPUS_PER_TASK \
* https://wiki.nci.nih.gov/display/TCGA/RNASeq
  --genomeDir STARindex \
* https://www.biostars.org/p/128132/
  --genomeFastaFiles /path/to/fasta.fa \
* [https://cran.r-project.org/web/packages/TCGAretriever/index.html TCGAretriever] from CRAN
  --sjdbGTFfile /path/to/genes.gtf \
* [https://www.bioconductor.org/packages/release/bioc/html/RTCGA.html RTCGA] package from Bioconductor.
  --sdjbOverhang 85
STAR --twopassMode Basic \
  --runThreadN $SLURM_CPUS_PER_TASK \
  --genomeDir STARindex \
  --outSAMmapqUnique 50 \
  --sjdbOverhang 85 \
  --readFilesIn r1.fastq.gz r2.fastq.gz \
  --readFilesCommand zcat \
  --outSAMtype BAM Unsorted \
  --outFileNamePrefix output/star
|-
| Subread
| subread-buildindex -o INDEX /path/to/fasta.fa
subread-align -T $SLURM_CPUS_PER_TASK -i INDEX -r R1.FASTQ.gz -R R2.FASTQ.gz -o output/alignment.sam
|-
| Hisat2
| hisat2-build -p $SLURM_CPUS_PER_TASK \
  /path/to/fasta.fa INDEX
hisat2 -p $SLURM_CPUS_PER_TASK -x INDEX \
  -1 R1.FASTQ.gz \
  -2 R2.FASTQ.gz -S output/alignment.sam
|}


The TCGA Data Portal does not host lower levels of sequence data. NCI's [https://cghub.ucsc.edu/ Cancer Genomics Hub (CGHub)] is the new secure repository for storing, cataloging, and accessing BAM files and metadata for sequencing data (need to 1. register from HHS to get an access 2. request access to the secure data set).
= What's special with rna-seq count data =
== Overdispersion ==
* http://genomicsclass.github.io/book/pages/rnaseq_gene_level.html
* [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081415 Dispersion Estimation and Its Effect on Test Performance in RNA-seq Data Analysis: A Simulation-Based Comparison of Methods] by Landau 2013.
* [https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-017-3809-0 Gene dispersion is the key determinant of the read count bias in differential expression analysis of RNA-seq data]


== UCI ==
== Normalization ==
* [http://archive.ics.uci.edu/ml/datasets.html?format=&task=&att=&area=life&numAtt=&numIns=&type=&sort=nameUp&view=table Machine learning repository] (not RNA-Seq)
* [http://www.hindawi.com/journals/bmri/2015/621690/ The Impact of Normalization Methods on RNA-Seq Data Analysis] by Zyprych-Walczak 2015. Two public datasets (Bodymap and Cheung) are available from [http://bowtie-bio.sourceforge.net/recount/ recount] website.
 
* [http://www.biomedcentral.com/1471-2105/11/94/ Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments] by Bullard 2010.
== ReCount ==
* [http://www.biomedcentral.com/1471-2105/16/347 Comparing the normalization methods for the differential analysis of Illumina high-throughput RNA-Seq data] by Peipei Li 2015.
* [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3229291/ ReCount: A multi-experiment resource of analysis-ready RNA-seq gene count datasets]
* [http://www.rna-seqblog.com/comparison-of-tmm-edger-rle-deseq2-and-mrn-normalization-methods/ Comparison of TMM (edgeR), RLE (DESeq2), and MRN Normalization Methods] by Maza E 2016
* http://bowtie-bio.sourceforge.net/recount/
* [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169594 A Hypothesis Testing Based Method for Normalization and Differential Expression Analysis of RNA-Seq Data] by Zhou 2017
* Two datasets were used in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631466/ Getting the most out of RNA-seq data analysis]
 
== Power Calculation ==
* [http://rnajournal.cshlp.org/content/20/11/1684 Power analysis and sample size estimation for RNA-Seq differential expression] Ching 2014
* [https://www.ncbi.nlm.nih.gov/pubmed?Db=pubmed&Cmd=ShowDetailView&TermToSearch=25273110 PROPER: comprehensive power evaluation for differential expression using RNA-seq] Wu 2015
* [http://journal.frontiersin.org/article/10.3389/fgene.2016.00225/full Extending the R Library PROPER to Enable Power Calculations for Isoform-Level Analysis with EBSeq] Gaye 2016
* [https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-017-1648-2 Power analysis for RNA-Seq differential expression studies] Yu 2017
 
== Phenotype Prediction ==
[http://www.biorxiv.org/content/early/2017/06/03/145656 Improving the value of public RNA-seq expression data by phenotype prediction]
 
== Clustering ==
[http://www.rna-seqblog.com/independent-component-analysis-ica-based-clustering-of-temporal-rna-seq-data/ ICAclust – Independent Component Analysis (ICA) based-clustering of temporal RNA-seq data]
 
== Correlation between RNA-Seq and microarrays ==
[http://www.sciencedirect.com/science/article/pii/S0378111917305796 Correlation between RNA-Seq and microarrays results using TCGA data]
 
== Techniques for characterizing RNA transcripts ==
qRT-PCR -> Microarray -> Nanostring -> RNA-Seq
 
[http://www.translationalres.com/article/S1931-5244(17)30229-3/fulltext Integrating RNA sequencing into neuro-oncology practice]
 
== Differential expression analysis ==
* [https://liorpachter.wordpress.com/2017/08/02/how-not-to-perform-a-differential-expression-analysis-or-science/ How not to perform a differential expression analysis (or science)]
* [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103207 A Comparative Study of Techniques for Differential Expression Analysis on RNA-Seq Data] (Zhang, 2014)
 
== Reproducible RNA-Seq analysis ==
* [https://f1000research.com/articles/6-586/v1  Arkas: Rapid reproducible RNAseq analysis]
* [https://f1000research.com/collections/containers Container Virtualization in Bioinformatics]
 
= Other RNA-Seq data =
 
== GEO ==
Go to http://www.ncbi.nlm.nih.gov/sites/entrez and select 'GEO Datasets' from the drop down menu, and use 'RNA-Seq' as your search term. http://www.ncbi.nlm.nih.gov/gds?term=rna-seq


= Other RNA-Seq tools =
* https://www.biostars.org/p/46059/
http://en.wikipedia.org/wiki/List_of_RNA-Seq_bioinformatics_tools
* https://www.biostars.org/p/52866/
 
=== GSE28666 ===
as used in DAFS paper. The timing is
* bowtie2-build 115 minutes
* fastq-dump 25 minutes per fastq (3.3 - 5.9GB fastq files)
* tophat2 90 minutes per fastq
* samtools 5 minutes
* htseq-count 13 minutes
 
=== GSE30567 ===
Used in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530905/ STAR] paper
 
=== GSE38886 ===
Used in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530905/ STAR] paper
 
=== GSE48215 & GSE48216 ===
[https://genomebiology.biomedcentral.com/articles/10.1186/gb-2013-14-10-r110 Modeling precision treatment of breast cancer]
 
GSE48215 is Whole exome Seq and GSE48216 is RNA-Seq
 
=== GSE51403 (full R script) ===
[http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51403 RNA-seq differential expression studies: more sequence, or more replication?]
 
An example of R code to download SRA files.
<pre>
        homo sapiens,
        Illumina HiSeq 2000.
        Question: what reference genome I should use?
        Ans1: hg18 was used.
              See http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1244821
              Or the paper.
 
        Ans2: GRCh38:
              Click 'BioProject' in http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51403
                then click 'Genome' in http://www.ncbi.nlm.nih.gov/bioproject/PRJNA222975
                But tophat website only provides GRCh37.
                https://genome.ucsc.edu/goldenPath/newsarch.html shows GRCh38 assembly is released
                in Dec/2013.
 
        38 fastq/SRR  (all are single lane)
        14 samples/GSM
 
        GSM#    SRX#    Sample          SRR#
      ---------------------------------------------------------------
Ctrl    1244822 365217  Ctrl_Rep7      1012952, 1012953, 1012954 (3 runs, 70M spots, 3.5G bases)
        1244821 365216  Ctrl_Rep6      949-951 (I just skip prefix '1012')
        1244820 365215  Ctrl_Rep5      946-948 (3 runs)
        1244819 365214  Ctrl_Rep4      943-945 (3 runs)  Control ethanol 24h
        1244818 365213  Ctrl_Rep3      940-942 (3 runs)
        1244817 365212  Ctrl_Rep2      937-939 (3 runs)
        1244816 365211  Ctrl_Rep1      934-936 (3 runs)  Control ethanol 24h
 
Trmnt  1244815 365210  E2_Rep7        931-933 (3 runs)  10nM E2 treatment for 24h
        1244814 365209  E2_Rep6        928-930 (3 runs)
        1244813 365208  E2_Rep5        925-927 (3 runs)
        1244812 365207  E2_Rep4        923-924 (2 runs)
        1244811 365206  E2_Rep3        921-922 (2 runs)
        1244810 365205  E2_Rep2        919-920 (2 runs)  10nM E2 treatment for 24h
        1244809 365204  E2_Rep1        917-918 (2 runs)  10nM E2 treatment for 24h
</pre>
<syntaxhighlight lang='rsplus'>
# Step 1. Download and convert data
 
library(SRAdb)
setwd("~/GSE51403")
if( ! file.exists('SRAmetadb.sqlite') ) {
        # sqlfile <- getSRAdbFile()
  sqlfile <- "~/Anders2013/SRAmetadb.sqlite"
} else {
sqlfile <- 'SRAmetadb.sqlite'
}
sra_con <- dbConnect(SQLite(),sqlfile)
fs <- listSRAfile("SRP031476", sra_con, fileType = "sra" )    # 38 files
# getSRAfile("SRP031476", sra_con, fileType = "sra" )          # starting to download
 
getSRAfile(fs$run[13], sra_con, fileType='sra') # SRR1012917.sra
getSRAfile(fs$run[30], sra_con, fileType='sra') # SRR1012918.sra
 
dbDisconnect( sra_con )
 
fs <- dir(pattern='sra')
# for(f in fs)  system(paste("fastq-dump --split-3", f)) # Single thread, Not efficient
scrp <- paste("/opt/RNA-Seq/bin/sratoolkit.2.3.5-2-ubuntu64/bin/fastq-dump --split-3", fs)
 
library(parallel)
cl <- makeCluster(getOption("cl.cores", 5))
# note that if something is wrong, we need to delete broken fastq first and then run again.
system.time(clusterApply(cl, scrp, function(x) system(x)))
stopCluster(cl)
 
# Step 2. Create samples object
samples <- data.frame(LibraryName = c(paste("Ctrl_Rep", 7:1, sep=''),
                                      paste("E2_Rep", 7:1, sep='')),
            LibraryLayout = rep("SINGLE", 14),
            fastq1=c(paste(paste("SRR", 1012952:1012954, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012949:1012951, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012946:1012948, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012943:1012945, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012940:1012942, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012937:1012939, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012934:1012936, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012931:1012933, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012928:1012930, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012925:1012927, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012923:1012924, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012921:1012922, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012919:1012920, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012917:1012918, ".fastq", sep=''), collapse=',')),
            fastq2=rep("", 14),
            countf=c(paste("Ctrl_Rep", 7:1, ".count", sep=''),
                      paste("E2_Rep", 7:1, ".count", sep='')))
# Step 3: run tophat2
i <- 10
system.time(paste("tophat2 -G genes.gtf -p 5 -o",
                  samples$LibraryName[i],
                  "genome",
                  samples$fastq1[i]))
# system("tophat2 -G genes.gtf -p 5 -o E2_Rep5 genome SRR1012925.fastq")
# system("tophat2 -G genes.gtf -p 5 -o E2_Rep5 genome SRR1012925.fastq,SRR1012926.fastq,SRR1012927.fastq")
for(i in c(1:14)) {
  system(paste("tophat2 -G genes.gtf -p 5 -o", samples$LibraryName[i], "genome", samples$fastq1[i]))
}
 
# Step 4. Organize, sort and index the BAM files and create SAM files.
for(i in seq_len(nrow(samples)) {
  lib = samples$LibraryName[i]
  ob = file.path(lib, "accepted_hits.bam")
  # sort by name, convert to SAM for htseq-count
  c1 <- paste0("samtools sort -n ", ob, " ", lib, "_sn")
  system(c1)
}
 
samtools sort -n Sample1/accepted_hits.bam Sample1_sn  # 283 seconds
# samtools sort Sample1/accepted_hits.bam Sample1_s
 
# Step 5. Count reads using htseq-count
htseq-count -f bam -s no -a 10 Sample1_sn.bam Mus_musculus.GRCm38.70.gtf > Sample1.count # 759 seconds
htseq-count -f bam -s no -a 10 Sample2_sn.bam Mus_musculus.GRCm38.70.gtf > Sample2.count
samples$countsf <- paste(samples$LibraryName, "count", sep=".")
gf <- "genes.gtf"
cmd <- paste0("htseq-count -s no -a 10 ", samples$LibraryName, "_sn.sam ", gf, " > ", samples$countf)
system.time(clusterApply(cl, cmd, function(x) system(x))) # 119 minutes
 
 
samplesDESeq = data.frame(shortname = c("Sample1", "Sample2"), countf=c("Sample1.count", "Sample2.count"),
                        condition = c("CTL", "KD"), LibraryLayout =c("SINGLE", "SINGLE"))
library("DESeq")
cds = newCountDataSetFromHTSeqCount(samplesDESeq)
cds = estimateSizeFactors(cds)
sizeFactors(cds)
</syntaxhighlight>
 
=== GSE37544 ===
[http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37544 RNA-seq analysis of differential gene expression in liver from lactating dairy cows divergent in negative energy balance]
 
=== GSE11045 ===
[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126545 Probe Region Expression Estimation for RNA-Seq Data for Improved Microarray Comparability]
 
=== [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37703 GSE37703/SRP012607] ===
Differential analysis of HOXA1 in adult cells at isoform resolution by RNA-Seq by Trapnell C, Hendrickson DG, Sauvageau M, Goff L et al. Nat Biotechnol 2013.
 
The <samples.txt> file required by BRB-SeqTools looks like
<pre>
LibraryName LibraryLayout fastq1 fastq2 ReadGroup SequencerManufacturer
LFB_scramble_repA PAIRED SRR493366_1.fastq SRR493366_2.fastq 1 illumina
LFB_scramble_repB PAIRED SRR493367_1.fastq SRR493367_2.fastq 2 illumina
LFB_scramble_repC PAIRED SRR493368_1.fastq SRR493368_2.fastq 3 illumina
LFB_HOXA1KD_repA PAIRED SRR493369_1.fastq SRR493369_2.fastq 4 illumina
LFB_HOXA1KD_repB PAIRED SRR493370_1.fastq SRR493370_2.fastq 5 illumina
LFB_HOXA1KD_repC PAIRED SRR493371_1.fastq SRR493371_2.fastq 6 illumina
</pre>
 
=== GSE37704 ===
http://www.gettinggeneticsdone.com/2015/12/tutorial-rna-seq-differential.html
 
=== GSE64570, GSE69244, GSE72165 ===
[http://f1000research.com/articles/4-1521/v1#DS3 Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences]
 
=== Ion Torrent ===
* [http://www.ncbi.nlm.nih.gov/geo/browse/?view=platforms&display=500&search=ion%20torrent&zsort=date GEO]
* [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE46876 GSE46876] from GPL17301 PGM (Homo sapiens) and GPL17303 Ion Torrent Proton (Homo sapiens). Unequal read length.
* [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE83668 GSE83668] from GPL17303 Ion Torrent Proton (Homo sapiens) and GPL11154 Illumina HiSeq 2000 (Homo sapiens)
* [https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR3214581 GSE79051/SRP071553] from GPL17303 Ion Torrent Proton (unequal read length)
 
== SRA ==
 
=== Pipeline ===
[https://www.biorxiv.org/content/early/2018/08/07/386441?rss=1 Extracting allelic read counts from 250,000 human sequencing runs in Sequence Read Archive] 2018. Code in [https://github.com/brianyiktaktsui/Skymap Github] and [https://www.synapse.org/#!Synapse:syn11415602/wiki/492470 Data].
 
=== SRA000299 ===
* [http://genome.cshlp.org/content/18/9/1509.full Paper]
* [http://davetang.org/muse/2012/05/09/getting-started-with-tophat/ Blog]
 
Note that one fastq file can represent several '''channels/lanes'''. The lane information can be extracted from fastq definition line (first line of each read). For example,
<pre>
@SRR002321.1 080226_CMLIVERKIDNEY_0007:2:1:115:885
</pre>
"SRR002321.1" is the short read archive name, where the .1 is the read number, "080226_CMLIVERKIDNEY_0007" should be the Machine name, which has been arbitrarily changed
* "2" is the Channel/lane number
* "1" is the Tile number
* "115" is the X position
* "885" is the Y position
The website also shows how to extract reads with the same '''run''' from fastq files.
 
In this example, there are
* two full sequencing runs (this info can be obtained from paper)
* 4 experiments (SRX000571, SRX000604, SRX000605, SRX000606)
* 6 runs (SRRR002321, SRR002323, SRR002322, SRR002320, SRR002325, SRR002324)
* 7 lanes (eg "080226_CMLIVERKIDNEY_0007:2") for each full sequencing run
 
=== SRA data linked from [http://athen.cecad.uni-koeln.de/quickngs/web/download/ QuickNGS] ===
Include Chip-Seq, RNA-Seq, WGS and miRNA-Seq. QuickNGS is a production environment for quick batch-wise analysis of Next-Generation Sequencing (NGS) data in high-throughput laboratories.
 
== [http://www.ebi.ac.uk/ena European Nucleotide Archive/ENA] ==
 
== [http://www.ddbj.nig.ac.jp/ DDBJ] ==
 
== ENCODE ==
http://www.ncbi.nlm.nih.gov/geo/info/ENCODE.html has a list of data with GSE number.
 
[https://www.encodeproject.org/pipelines/ Data Processing Pipelines]
 
== [http://www.1000genomes.org 1000 genomes] ==
 
== Human BodyMap 2.0 data from Illumina ==
* http://www.ensembl.info/blog/2011/05/24/human-bodymap-2-0-data-from-illumina/
* https://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise. Galaxy needs to install hg19 genome before we can use tophat or bowtie. See [https://wiki.galaxyproject.org/Admin/DataPreparation?action=show&redirect=Admin%2FNGS+Local+Setup Galaxy/Wiki/Admin/Data Preparation] page. Genome can be downloaded from [http://tophat.cbcb.umd.edu/igenomes.shtml tophat] webpage. It is 20GB for hg19! We can also download just chromosome 19 fasta file [http://hgdownload.cse.ucsc.edu/goldenPath/hg19/chromosomes/ chr19.fa.gz] from [http://hgdownload.cse.ucsc.edu/downloads.html UCSC genomes FTP server] and it is only 19MB. We can then create reference index files (.bt2) by using bowtie2-build command. If we use Galaxy, we don't need to create reference index files.
* [http://www.biomedcentral.com/1471-2164/16/97 Impact of gene annotation on RNA-Seq data analysis]
 
== [https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp TCGA/The Cancer Genome Atlas] ==
* [http://gdac.broadinstitute.org/ Broad Institute TCGA GDAC]
* https://wiki.nci.nih.gov/display/TCGA/RNASeq
* https://www.biostars.org/p/128132/
* [https://cran.r-project.org/web/packages/TCGAretriever/index.html TCGAretriever] from CRAN
* [https://www.bioconductor.org/packages/release/bioc/html/RTCGA.html RTCGA] package from Bioconductor.
** [http://www.bioconductor.org/packages/release/data/experiment/html/RTCGA.clinical.html RTCGA.clinical] - Clinical datasets from The Cancer Genome Atlas Project
** [https://rtcga.github.io/RTCGA/reference/survivalTCGA.html Extract Survival Information from Datasets Included in RTCGA.clinical and RTCGA.clinical.20160128 Packages]
** https://rtcga.github.io/RTCGA/reference/expressionsTCGA.html
<pre>
> if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
> BiocManager::install("RTCGA.clinical")
> library(RTCGA.clinical)
 
# Available cohorts
> checkTCGA('Dates')
[1] "2011-10-26" "2011-11-15" "2011-11-28" "2011-12-06" "2011-12-30"
[6] "2012-01-10" "2012-01-24" "2012-02-17" "2012-03-06" "2012-03-21"
[11] "2012-04-12" "2012-04-25" "2012-05-15" "2012-05-25" "2012-06-06"
[16] "2012-06-23" "2012-07-07" "2012-07-25" "2012-08-04" "2012-08-25"
[21] "2012-09-13" "2012-10-04" "2012-10-18" "2012-10-20" "2012-10-24"
[26] "2012-11-02" "2012-11-14" "2012-12-06" "2012-12-21" "2013-01-16"
[31] "2013-02-03" "2013-02-22" "2013-03-09" "2013-03-26" "2013-04-06"
[36] "2013-04-21" "2013-05-08" "2013-05-23" "2013-06-06" "2013-06-23"
[41] "2013-07-15" "2013-08-09" "2013-09-23" "2013-10-10" "2013-11-14"
[46] "2013-12-10" "2014-01-15" "2014-02-15" "2014-03-16" "2014-04-16"
[51] "2014-05-18" "2014-06-14" "2014-07-15" "2014-09-02" "2014-10-17"
[56] "2014-12-06" "2015-02-02" "2015-02-04" "2015-04-02" "2015-06-01"
[61] "2015-08-21" "2015-11-01" "2016-01-28"
# Available cohorts
> (cohorts <- infoTCGA() %>% rownames() %>% sub("-counts", "", x=.))
 
[1] "ACC"      "BLCA"    "BRCA"    "CESC"    "CHOL"    "COAD"   
[7] "COADREAD" "DLBC"    "ESCA"    "FPPP"    "GBM"      "GBMLGG" 
[13] "HNSC"    "KICH"    "KIPAN"    "KIRC"    "KIRP"    "LAML"   
[19] "LGG"      "LIHC"    "LUAD"    "LUSC"    "MESO"    "OV"     
[25] "PAAD"    "PCPG"    "PRAD"    "READ"    "SARC"    "SKCM"   
[31] "STAD"    "STES"    "TGCT"    "THCA"    "THYM"    "UCEC"   
[37] "UCS"      "UVM"
> dim(BRCA.clinical)
[1] 1098 3703
> dim(OV.clinical) # one row per sample
[1]  591 3626
> dim(LUAD.clinical)
[1]  522 3046
> OV.survInfo <- survivalTCGA(OV.clinical, extract.cols = "admin.disease_code")
> dim(OV.survInfo)  # 576 x 4
> plot(survfit(Surv(times, patient.vital_status) ~ 1,
      data = OV.survInfo), conf.int=F, mark.time=TRUE)
 
> BiocManager::install("RTCGA.mRNA")
> data(package = "RTCGA.mRNA")
> data(OV.mRNA, package = "RTCGA.mRNA")
> dim(OV.mRNA)  # one row per sample
[1]  561 17815
> OV.mRNA[1:2, 1:5]
          bcr_patient_barcode      ELMO2  CREB3L1    RPS11  PNMA1
1 TCGA-01-0628-11A-01R-0363-07 -0.24800000 -0.14150 1.370625 1.08775
2 TCGA-01-0639-11A-01R-0363-07  0.09508333 -0.04650 0.982875 1.38725
> OV.clinical[1:2, 18]
[1] "tcga-04-1331" "tcga-04-1332"
 
> match(substr(OV.mRNA[, 1], 6, 12), substr(OV.clinical[, 18], 6, 12)) %>%
  na.omit() %>% length()
[1] 546
</pre>
 
The TCGA Data Portal does not host lower levels of sequence data. NCI's [https://cghub.ucsc.edu/ Cancer Genomics Hub (CGHub)] is the new secure repository for storing, cataloging, and accessing BAM files and metadata for sequencing data (need to 1. register from HHS to get an access 2. request access to the secure data set).
 
== UCI ==
* [http://archive.ics.uci.edu/ml/datasets.html?format=&task=&att=&area=life&numAtt=&numIns=&type=&sort=nameUp&view=table Machine learning repository] (not RNA-Seq)
 
== ReCount ==
* [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3229291/ ReCount: A multi-experiment resource of analysis-ready RNA-seq gene count datasets]
* http://bowtie-bio.sourceforge.net/recount/
* Two datasets were used in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631466/ Getting the most out of RNA-seq data analysis]
 
= Other RNA-Seq tools =
http://en.wikipedia.org/wiki/List_of_RNA-Seq_bioinformatics_tools


== [https://wiki.galaxyproject.org/Admin/GetGalaxy Galaxy] ==
== [https://wiki.galaxyproject.org/Admin/GetGalaxy Galaxy] ==
The first 3 lines will install galaxy on local computer. The last command will start Galaxy. PS. It will take a while to start the first time.
The first 3 lines will install galaxy on local computer. The last command will start Galaxy. PS. It will take a while to start the first time.
<pre>
<pre>
hg clone https://bitbucket.org/galaxy/galaxy-dist/
hg clone https://bitbucket.org/galaxy/galaxy-dist/
cd galaxy-dist
cd galaxy-dist
hg update stable
hg update stable
sh run.sh
sh run.sh
</pre>
</pre>
A screenshot of the available tools is given below. Strangely, I did not find "NGS: RNA Analysis" in the NGS: ToolBox on left hand side.
A screenshot of the available tools is given below. Strangely, I did not find "NGS: RNA Analysis" in the NGS: ToolBox on left hand side.
 
 
[[File:GalaxyInitial.png|200px]]
[[File:GalaxyInitial.png|200px]]
 
We cannot upload > 2GB fastq data. For such large dataset (we have one 3.3 GB fastq in this case), Galaxy asks to use http or ftp to load the data. I install Apache in Ubuntu and put this fastq file there for upload.
 
Galaxy does not include bowtie, tophat, .... by default. See [http://seqanswers.com/forums/showthread.php?t=10315 this post] and [https://wiki.galaxyproject.org/Admin/DataPreparation?action=show&redirect=Admin%2FNGS+Local+Setup Galaxy wiki] about the installation. Another option is to modify line 614 of 'universe_wsgl.ini' file and enable ourselves as adminstrator. Also modify line 146 to add a path for installing tools through Galaxy Admin page.
 
If you submit a lot of jobs (eg each tophat2 generates 5 jobs) some of jobs cannot be run immediately. In my experience, there is a 25 jobs cap. Running jobs has a yellow background color. Waiting for run jobs has a gray background color.
 
Some helpful information
* [https://galaxyproject.org/virtual-appliances/ Virtual Appliances]
* [https://github.com/bgruening/docker-galaxy-stable Galaxy Docker Image]
* [http://seqanswers.com/forums/showthread.php?t=23629 Quick & dirty Galaxy installation in a virtual machine]
* [https://biostar.usegalaxy.org/p/1860/ Downloadable Galaxy Virtual Machine In Vmware]. The virtual machine is not there anymore but the [https://wiki.nbic.nl/index.php/Galaxy_VM documentation] is still there.
* https://wiki.galaxyproject.org/CitingGalaxy Citation
* https://wiki.galaxyproject.org/Learn Search rna-seq or NGS as keywords.
* https://biostar.usegalaxy.org/
* [https://sites.google.com/site/princetonhtseq/tutorials/rna-seq Galaxy & DESeq2]
* [https://youtu.be/rVYzMWWRMN8 Galaxy Demo -- RNAseq Pipeline as Example] from BioIT Core.
 
== Cufflink -> Cuffcompare or Cuffmerge -> Cuffdiff ==
* http://cole-trapnell-lab.github.io/cufflinks/
* [http://dingo.ucsf.edu/twiki/bin/view/Cores/BioinformaticsCore/RnaSeqAnalysis UCSF] wiki.
* [http://ged.msu.edu/angus/tutorials-2013/DGE_analysis_with_MISO_cuffdiff.html Michigan State University]
* http://seqanswers.com/forums/showthread.php?t=9753
* https://docs.uabgrid.uab.edu/wiki/UAB_Galaxy_RNA_Seq_Step_by_Step_Tutorial#Cufflinks_inputs
* https://sites.google.com/site/princetonhtseq/tutorials/rna-seq
* http://www.genomecenter.ucdavis.edu/resources/instructional-videos
 
== Forum ==
* [http://seqanswers.com/ Seqanswers.com]
* [https://www.biostars.org/ Biostars.org]
* [https://biostar.usegalaxy.org/ biostar.usegalaxy.org]


We cannot upload > 2GB fastq data. For such large dataset (we have one 3.3 GB fastq in this case), Galaxy asks to use http or ftp to load the data. I install Apache in Ubuntu and put this fastq file there for upload.
== recount2 ==
A multi-experiment resource of analysis-ready RNA-seq gene and exon count datasets
https://jhubiostatistics.shinyapps.io/recount/


Galaxy does not include bowtie, tophat, .... by default. See [http://seqanswers.com/forums/showthread.php?t=10315 this post] and [https://wiki.galaxyproject.org/Admin/DataPreparation?action=show&redirect=Admin%2FNGS+Local+Setup Galaxy wiki] about the installation. Another option is to modify line 614 of 'universe_wsgl.ini' file and enable ourselves as adminstrator. Also modify line 146 to add a path for installing tools through Galaxy Admin page.
== recount3 ==
* https://rna.recount.bio/
* [https://support.bioconductor.org/p/9153662/ Zero counts for all samples for all genes downloaded from recount3]


If you submit a lot of jobs (eg each tophat2 generates 5 jobs) some of jobs cannot be run immediately. In my experience, there is a 25 jobs cap. Running jobs has a yellow background color. Waiting for run jobs has a gray background color.
== RNACocktail ==
[https://bioinform.github.io/rnacocktail/ A comprehensive framework for accurate and efficient RNA-Seq analysis]


Some helpful information
== [http://www.biorxiv.org/content/biorxiv/early/2017/09/14/189092.full.pdf Massive Mining of Publicly Available RNA-seq Data from Human and Mouse] ==
* [https://biostar.usegalaxy.org/p/1860/ Downloadable Galaxy Virtual Machine In Vmware]. The virtual machine is not there anymore but the [https://wiki.nbic.nl/index.php/Galaxy_VM documentation] is still there.
* [http://amp.pharm.mssm.edu/archs4/ ARCHS4: All RNA and ChIP-Seq Sample and Signature Search]
* https://wiki.galaxyproject.org/CitingGalaxy Citation
* [https://youtu.be/xw8vh_sYJ4s ARCHS4 tutorial] (youtube). The chrome extension did not work on GSE26880 (still no ARCHS4 icon on the GEO webpage after restart Chrome. Click the series matrix file link, it brings to a page to download 3 GPL series matrix. But the series matrix files still don't contain the expression)
* https://wiki.galaxyproject.org/Learn Search rna-seq or NGS as keywords.
* https://biostar.usegalaxy.org/
* [https://sites.google.com/site/princetonhtseq/tutorials/rna-seq Galaxy & DESeq2]
* [https://youtu.be/rVYzMWWRMN8 Galaxy Demo -- RNAseq Pipeline as Example] from BioIT Core.


== Cufflink -> Cuffcompare or Cuffmerge -> Cuffdiff ==
= Misc =
* http://cole-trapnell-lab.github.io/cufflinks/
== Markets ==
* [http://dingo.ucsf.edu/twiki/bin/view/Cores/BioinformaticsCore/RnaSeqAnalysis UCSF] wiki.
[http://www.rna-seqblog.com/global-and-united-states-ngs-based-rna-seq-market-2017/ Global And United States NGS-based RNA-seq Market 2017]
* [http://ged.msu.edu/angus/tutorials-2013/DGE_analysis_with_MISO_cuffdiff.html Michigan State University]
* http://seqanswers.com/forums/showthread.php?t=9753
* https://docs.uabgrid.uab.edu/wiki/UAB_Galaxy_RNA_Seq_Step_by_Step_Tutorial#Cufflinks_inputs
* https://sites.google.com/site/princetonhtseq/tutorials/rna-seq
* http://www.genomecenter.ucdavis.edu/resources/instructional-videos


== Forum ==
== Tutorial/Primer ==
* [http://seqanswers.com/ Seqanswers.com]
[http://onlinelibrary.wiley.com/doi/10.1111/cts.12511/abstract Whole Transcriptome Profiling: An RNA-Seq Primer and Implications for Pharmacogenomics Research]
* [https://www.biostars.org/ Biostars.org]
* [https://biostar.usegalaxy.org/ biostar.usegalaxy.org]

Latest revision as of 15:31, 8 October 2024

The data is used in the paper "Count-based differential ....." by Anders et al 2013. The paper has been cited 8136 times according to scholar.google.com and 4330 times according to PubMed on Feb 7, 2019.

This page focuses on RNA-Seq. For DNA-Seq see the Genome page.

Required tools

The version number indicated below is the one I use. It may be updated when you are ready to download that.

  • bowtie (bowtie2-2.1.0-linux-x86_64.zip): Mac, Linux, Windows binaries
  • samtools (samtools-0.1.19.tar.bz2): require GCC to compile (build-essential) & zlib1g-dev & libncurses5-dev packages. wikipedia.
  • tophat (tophat-2.0.10.Linux_x86_64.tar.gz): Linux and Mac binaries. Mailing list is on Tuxedo Tools Users Google Group.
  • sra toolkit (sratoolkit.2.3.4-2-ubuntu64.tar.gz): Mac, Ubuntu & CentOS binaries
  • HTseq (HTSeq-0.6.1.tar.gz): require Python 2 and pysam package
  • IGV (IGV_2.3.26.zip): require Java and registration to download the program
  • FastQC (fastqc_v0.10.1.zip): require Java (jdk is needed, jre is not enough)
  • FastX (fastx_toolkit_0.0.13_binaries_Linux_2.6_amd64.tar.bz2): require libgtextutils-0.6

Note

  • For alignment, STAR is another choice.
  • For trimmer, trimmomatic is another choice. For example, we can use it to remove low quality reads (<30).
java -jar trimmomatic-0.36.jar -phred33 -threads 8 file1.fastq.gz file2.fastq.gz -baseout file.fastq.gz AVGQUAL:30

java -jar trimmomatic-0.36.jar -phred33 -threads 8 file1.fastq.gz file2.fastq.gz -baseout file.fastq.gz HEADCROP:5 MINLEN:50 AVGQUAL:20

Installation

If binary executables are available, we don't need to do anything special except adding the path containing the binary to the PATH variable.

If only source code is available, make sure the required tool GCC is installed (Under Ubuntu, we can use sudo apt-get install build-essential). Then we can compile and then install the program by using ./configure, make and make install.

In this exercise, all source code or binary programs were extracted to $HOME/binary/ directory (Use mkdir ~/binary to create this new directory) where each program has its own directory.

To use a shell script to install every thing, try the following. 

cd; wget -N https://www.dropbox.com/s/ip1jiarxhq0dq91/install.sh; chmod +x install.sh; sudo ./install.sh; rm install.sh

Change PATH variable

Open ~/.basrhc file using any text editor (such as nano or vi) and add the path containing the binary of each program to PATH variable. 

tail ~/.bashrc
export PATH=$PATH:~/binary/bowtie2-2.1.0/
export PATH=$PATH:~/binary/tophat-2.0.10.Linux_x86_64/
export PATH=$PATH:~/binary/samtools-0.1.19/
export PATH=$PATH:~/binary/samtools-0.1.19/bcftools/
export PATH=$PATH:~/binary/samtools-0.1.19/misc/
export PATH=$PATH:~/binary/sratoolkit.2.3.4-2-ubuntu64/bin
export PATH=$PATH:~/binary/HTSeq-0.6.1/build/scripts-2.7
export PATH=$PATH:~/binary/IGV_2.3.26/
export PATH=$PATH:~/binary/FastQC/
export PATH=$PATH:~/binary/fastx/bin

If we want to make these tools available for ALL users (ie system wide environment), we need to put these lines on /etc/profile file.

HTSeq and pysam

HTSeq requires Python 2 and pysam package. For pysam package, we have two ways to install it

1. Use pip 

sudo apt-get install python-pip
# sudo -H pip install pysam
sudo -H pip install pysam==0.10.0  # lock the version

The "-H" flag is used to suppress a warning message.

The latest version of pysam may fail so it is not good to use the command pip install pysam. 

$ python
>>> import pysam
Traceback (most recent call last):
  File "<stdin>", line 1, in <module>
  File "/Library/Python/2.7/site-packages/pysam/__init__.py", line 5, in <module>
    from pysam.libchtslib import *
  File "pysam/libchtslib.pyx", line 1, in init pysam.libchtslib (pysam/libchtslib.c:12515)
ImportError: dlopen(/Library/Python/2.7/site-packages/pysam/libcutils.so, 2): Library not loaded: @rpath/pysam-0.11.2.2-py2.7-macosx-10.12-intel.egg/pysam/libcsamtools.so
  Referenced from: /Library/Python/2.7/site-packages/pysam/libcutils.so
  Reason: image not found

2. Use python to build/install. 

wget https://pypi.python.org/packages/de/03/02934438b204565bc5231f38a11da840a3c3e4b2beac8c8770d675770668/pysam-0.9.1.4.tar.gz
tar xzvf pysam-0.9.1.4.tar.gz
sudo apt-get -y install zlib1g-dev
sudo apt-get install python-setuptools
python setup.py build
sudo python setup.py install 
# Installed /usr/local/lib/python2.7/dist-packages/pysam-0.9.1.4-py2.7-linux-x86_64.egg

Data directory

Download <nprot.2013.099-S1.zip> from the paper's web site and extract it to ~/Anders2013 directory. 

brb@brbweb4:~$ mkdir Anders2013
brb@brbweb4:~$ cd Anders2013
brb@brbweb4:~/Anders2013$ wget http://www.nature.com/nprot/journal/v8/n9/extref/nprot.2013.099-S1.zip
brb@brbweb4:~/Anders2013$ unzip nprot.2013.099-S1.zip
brb@brbweb4:~/Anders2013$ ls
CG8144_RNAi-1.count  counts.csv             README.txt      Untreated-1.count  Untreated-6.count
CG8144_RNAi-3.count  __MACOSX               samples.csv     Untreated-3.count
CG8144_RNAi-4.count  nprot.2013.099-S1.zip  SraRunInfo.csv  Untreated-4.count

The raw data is from GSE18508 'modENCODE Drosophila RNA Binding Protein RNAi RNA-Seq Studies'.

Subdirectory

During running tophat program, several subdirectories will be generated. Each subdirectory will contain accepted_hits.bam, junctions.bed, insertions.bed, deletions.bed and other files/sub-subdirectories.

Once samtools program was run, .bam (and .bai) files will be created under Anders2013 directory.

Pipeline

The following summary is extracted from http://www.bioconductor.org/help/course-materials/2013/CSAMA2013/tuesday/afternoon/DESeq_protocol.pdf. It is the same as Anders 2013 paper but the paper requires an access permission.

It would be interesting to monitor the disk space usage before, during and after the execution.

Other pipelines:

Download example data (22 files in SRA format)

Download Supplement file and read <SraRunInfo.csv> in R. We will choose a subset of data based on "LibraryName" column. 

sri = read.csv("SraRunInfo.csv", stringsAsFactors=FALSE)
keep = grep("CG8144|Untreated-",sri$LibraryName)
sri = sri[keep,]
fs = basename(sri$download_path)
for(i in 1:nrow(sri))
 download.file(sri$download_path[i], fs[i])
names(sri)
[1] "Run"              "ReleaseDate"      "spots"            "bases"           
[5] "avgLength"        "size_MB"          "AssemblyName"     "download_path"   
[9] "Experiment"       "LibraryName"      "LibraryStrategy"  "LibrarySelection"
[13] "LibrarySource"    "LibraryLayout"    "InsertSize"       "Platform"        
[17] "Model"            "SRAStudy"         "BioProject"       "Study_Pubmed_id"
[21] "ProjectID"        "Sample"           "BioSample"        "TaxID"           
[25] "ScientificName"   "SampleName"       "Submission"       "Consent"        
apply(sri,2, function(x) length(unique(x)))
            Run      ReleaseDate            spots            bases
             22               22               22               22
      avgLength          size_MB     AssemblyName    download_path
              5               22                1               22
     Experiment      LibraryName  LibraryStrategy LibrarySelection
              7                7                1                1
  LibrarySource    LibraryLayout       InsertSize         Platform
              1                2                2                1
          Model         SRAStudy       BioProject  Study_Pubmed_id
              1                1                1                1
      ProjectID           Sample        BioSample            TaxID
              1                7                1                1
 ScientificName       SampleName       Submission          Consent
              1                1                1                1
sri[1:2,]
       Run         ReleaseDate   spots     bases avgLength size_MB
1 SRR031714 2010-01-15 10:42:00 5327425 394229450        74     396
2 SRR031715 2010-01-21 14:24:18 5248396 388381304        74     390
 AssemblyName
1           NA
2           NA
                                                                                         download_path
1 ftp://ftp-private.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR031/SRR031714/SRR031714.sra
2 ftp://ftp-private.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR031/SRR031715/SRR031715.sra
 Experiment LibraryName LibraryStrategy LibrarySelection  LibrarySource
1  SRX014459 Untreated-3         RNA-Seq             cDNA TRANSCRIPTOMIC
2  SRX014459 Untreated-3         RNA-Seq             cDNA TRANSCRIPTOMIC
 LibraryLayout InsertSize Platform                       Model  SRAStudy
1        PAIRED        200 ILLUMINA Illumina Genome Analyzer II SRP001537
2        PAIRED        200 ILLUMINA Illumina Genome Analyzer II SRP001537
                     BioProject Study_Pubmed_id ProjectID    Sample BioSample
1 GEO Series accession: GSE18508        20921232    168994 SRS008447        NA
2 GEO Series accession: GSE18508        20921232    168994 SRS008447        NA
 TaxID          ScientificName              SampleName Submission Consent
1  7227 Drosophila melanogaster Drosophila melanogaster  SRA010243  public
2  7227 Drosophila melanogaster Drosophila melanogaster  SRA010243  public

Convert SRA to FASTQ (sra -> fastq)

(Update) fasterq-dump command. Note there is no --gzip option as in fastq-dump. Use gzip to do that.

This takes a long time and it is not to run in parallel by default. At the end of execution, there are 30 fastq files because 8 SRA file are pair end. 

stopifnot( all(file.exists(fs)) ) # assure FTP download was successful
for(f in fs) {
cmd = paste("fastq-dump --split-3", f)
cat(cmd,"\n")
system(cmd) # invoke command
}

fastq-dump --split-3 SRR031714.sra
Read 5327425 spots for SRR031714.sra
Written 5327425 spots for SRR031714.sra
fastq-dump --split-3 SRR031715.sra
Read 5248396 spots for SRR031715.sra
Written 5248396 spots for SRR031715.sra
...

Note that '--split-3' option will splits mate-pair reads into separate files (First biological reads satisfying dumping conditions are placed in files *_1.fastq and *_2.fastq If only one biological read is present it is placed in *.fastq Biological reads and above are ignored). See this post. 

brb@brbweb4:~/Anders2013$ ls -lh *.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 12:47 SRR031708.fastq
-rw-r--r-- 1 brb brb 812M Feb 10 12:50 SRR031709.fastq
-rw-r--r-- 1 brb brb 1.2G Feb 10 12:56 SRR031710.fastq
-rw-r--r-- 1 brb brb 1.2G Feb 10 13:02 SRR031711.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 13:08 SRR031712.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 13:14 SRR031713.fastq
-rw-r--r-- 1 brb brb 1.1G Feb 10 11:48 SRR031714_1.fastq
-rw-r--r-- 1 brb brb 1.1G Feb 10 11:48 SRR031714_2.fastq
-rw-r--r-- 1 brb brb 1.1G Feb 10 11:54 SRR031715_1.fastq
-rw-r--r-- 1 brb brb 1.1G Feb 10 11:54 SRR031715_2.fastq
-rw-r--r-- 1 brb brb 1.2G Feb 10 12:02 SRR031716_1.fastq
-rw-r--r-- 1 brb brb 1.2G Feb 10 12:02 SRR031716_2.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 12:10 SRR031717_1.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 12:10 SRR031717_2.fastq
-rw-r--r-- 1 brb brb 1.4G Feb 10 13:21 SRR031718.fastq
-rw-r--r-- 1 brb brb 927M Feb 10 13:25 SRR031719.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 13:32 SRR031720.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 13:39 SRR031721.fastq
-rw-r--r-- 1 brb brb 1.2G Feb 10 13:45 SRR031722.fastq
-rw-r--r-- 1 brb brb 842M Feb 10 13:49 SRR031723.fastq
-rw-r--r-- 1 brb brb 1.2G Feb 10 12:17 SRR031724_1.fastq
-rw-r--r-- 1 brb brb 1.2G Feb 10 12:17 SRR031724_2.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 12:25 SRR031725_1.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 12:25 SRR031725_2.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 12:33 SRR031726_1.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 12:33 SRR031726_2.fastq
-rw-r--r-- 1 brb brb 1.2G Feb 10 12:41 SRR031727_1.fastq
-rw-r--r-- 1 brb brb 1.2G Feb 10 12:41 SRR031727_2.fastq
-rw-r--r-- 1 brb brb 1.3G Feb 10 13:54 SRR031728.fastq
-rw-r--r-- 1 brb brb 3.2G Feb 10 14:07 SRR031729.fastq

FASTQ PAIR

FASTQ PAIR Rewrite paired end fastq files to make sure that all reads have a mate and to separate out singletons

Access quality control through ShortRead or FastQC

library("ShortRead")
fqQC = qa(dirPath=".", pattern=".fastq$", type="fastq")
report(fqQC, type="html", dest="fastqQAreport")

Use a web browser to inspect the generated HTML file (here, stored in the \fastqQAreport" directory) with the quality-assessment report.

FastQC can be run either interactively or non-interactive. For example 

/opt/RNA-Seq/bin/FastQC/fastqc ~/GSE11209/SRR002058.fastq --outdir=/home/brb/GSE11209/fastQC

will create a zip file and an unzipped directory under /home/brb/GSE11209/fastQC.

FastX_trimmer is a better choice. The syntax is 

fastx_trimmer -f N -l N -i INPUT -o OUTPUT -v -Q33

The last parameter -Q33 is necessary for Illumina encoding quality score. If we omit it, we will get an error fastx_trimmer: Invalid quality score value (char '#' ord 35 quality value -29) on line 4

Read quality issues

http://training.bioinformatics.ucdavis.edu/docs/2012/05/RNA/qa-and-i.html

  1. Per base sequence quality (by position)
  2. Per sequence quality scores: see if a subset of sequences have universally low quality values.
  3. Per base sequence content
  4. Per sequence GC content
  5. Per base N content
  6. Sequence Length Distribution
  7. Sequence Duplication Levels
  8. Overpresented sequences
  9. Kmer Content

Encoding

Download reference genome (for Tophat)

wget ftp://ftp.ensembl.org/pub/release-70/fasta/drosophila_melanogaster/dna/Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa.gz
gunzip Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa.gz

Note that bowtie2/tophat2 indices for many commonly used reference genomes can be downloaded directly from https://ccb.jhu.edu/software/tophat/igenomes.shtml. Actually the link https://support.illumina.com/sequencing/sequencing_software/igenome.html from illumina is more complete.

For human species, it has different assemblies

  • Ensembl - GRCh37 (equivalent to hg19)
  • NCBI - GRCh38, build37.1, build37.2, build36.3 (equivalent to hg18)
  • UCSC - hg38, hg19, hg18
Source
Ensembl GRCh37
NCBI GRCh38 build37 build36
UCSC hg38 hg19 hg18

Structure of downloaded reference genome from Tophat

Downloaded genome from tophat takes a long time (20GB for human). Using bowtie2-build program which can takes an enormous time to run. 

$ tar xzvf Homo_sapiens_Ensembl_GRCh37.tar.gz
$ tree -L 4 Homo_sapiens
Homo_sapiens
└── Ensembl
    └── GRCh37
        ├── Annotation
        │   ├── Archives             <==== gtf file
        │   ├── Genes -> Archives/archive-current/Genes
        │   ├── README.txt -> Archives/archive-current/README.txt
        │   ├── SmallRNA -> Archives/archive-current/SmallRNA
        │   └── Variation -> Archives/archive-current/Variation
        ├── GenomeStudio
        │   ├── Archives
        │   ├── Homo_sapiens -> Archives/archive-2012-03-09-04-49-46/Homo_sapiens
        │   └── README.txt
        └── Sequence
            ├── AbundantSequences
            ├── Bowtie2Index         <==== bt2 files 
            ├── BowtieIndex
            ├── BWAIndex
            ├── Chromosomes
            ├── Squashed-Homo_sapiens-Ensembl-GRCh37
            └── WholeGenomeFasta     <==== fa file

18 directories, 2 files
$ ls -l Homo_sapiens/Ensembl/GRCh37/Annotation/Archives/archive-2013-03-06-14-23-04/Genes
total 639136
-rwxrwxr-x 1 brb brb       302 Mar 18  2013 ChromInfo.txt
-rwxrwxr-x 1 brb brb 603096976 Mar 18  2013 genes.gtf
-rwxrwxr-x 1 brb brb   8236175 Mar 18  2013 refFlat.txt.gz
-rwxrwxr-x 1 brb brb  43130092 Mar 18  2013 refGene.txt

A list of files in <Homo_sapiens_UCSC_hg18.tar.gz> can be found on (File:Ucsc hg18.txt) or <Homo_sapiens_UCSC_hg19.tar.gz> can be found on github.

We don't need to extract the whole tarball. We just need to extract some files we need. But how do we know the locations of files we need? Use tar -tzvf XXX.tar.gz to find out. 

$ tar -xzvf Homo_sapiens_UCSC_hg18.tar.gz Homo_sapiens/UCSC/hg18/Annotation/Archives/archive-2014-06-02-13-47-56/Genes/genes.gtf
Homo_sapiens/UCSC/hg18/Annotation/Archives/archive-2014-06-02-13-47-56/Genes/genes.gtf
$ tar -xzvf Homo_sapiens_UCSC_hg18.tar.gz Homo_sapiens/UCSC/hg18/Sequence/Bowtie2Index/
Homo_sapiens/UCSC/hg18/Sequence/Bowtie2Index/
Homo_sapiens/UCSC/hg18/Sequence/Bowtie2Index/genome.3.bt2
Homo_sapiens/UCSC/hg18/Sequence/Bowtie2Index/genome.1.bt2
Homo_sapiens/UCSC/hg18/Sequence/Bowtie2Index/genome.rev.2.bt2
Homo_sapiens/UCSC/hg18/Sequence/Bowtie2Index/genome.rev.1.bt2
Homo_sapiens/UCSC/hg18/Sequence/Bowtie2Index/genome.4.bt2
Homo_sapiens/UCSC/hg18/Sequence/Bowtie2Index/genome.2.bt2
Homo_sapiens/UCSC/hg18/Sequence/Bowtie2Index/genome.fa
$ tar -xzvf Homo_sapiens_UCSC_hg18.tar.gz Homo_sapiens/UCSC/hg18/Sequence/WholeGenomeFasta/genome.fa
Homo_sapiens/UCSC/hg18/Sequence/WholeGenomeFasta/genome.fa
$ tree -L 7 Homo_sapiens
Homo_sapiens
    └── hg18
        ├── Annotation
        │   └── Archives
        │       └── archive-2014-06-02-13-47-56
        │           └── Genes
        │               └── genes.gtf                             <= gtf file
        └── Sequence
            ├── Bowtie2Index
            │   ├── genome.1.bt2                                  <= bt2 file
            │   ├── genome.2.bt2
            │   ├── genome.3.bt2
            │   ├── genome.4.bt2
            │   ├── genome.fa -> ../WholeGenomeFasta/genome.fa    <= fake fa file (symbolic link)
            │   ├── genome.rev.1.bt2
            │   └── genome.rev.2.bt2
            └── WholeGenomeFasta
                └── genome.fa                                     <= fa file

Download GTF (gene transfer format) file (for Tophat)

The compressed file is 77.6 MB size. 

wget ftp://ftp.ensembl.org/pub/release-70/gtf/drosophila_melanogaster/Drosophila_melanogaster.BDGP5.70.gtf.gz
gunzip Drosophila_melanogaster.BDGP5.70.gtf.gz

CRITICAL: Make sure that the gene annotation uses the same coordinate system as the reference FASTA le. Here, both les use BDGP5 (i. e., release 5 of the assembly provided by the Berkeley Drosophila Genome Project), as is apparent from the le names. To be on the safe side here, we recommend to always download the FASTA reference sequence and the GTF annotation data from the same resource provider.

  • Ensembl.org provides both fasta and gtf files together. It's useful when we want to use Galaxy.
  • Broad also provides a link to download gtf and fasta file.

Preprocess reference FASTA files into an index (fasta -> bt2)

Alternatively, pre-built indices for many commonly-used genomes are available from http://tophat.cbcb.umd.edu/igenomes.html <Drosophila_melanogaster_Ensembl_BDGP5.tar.gz> contains genome.1.bt2, genome.2.bt2, genome.3.bt2, genome.fa, genome.rev.1.bt2, and genome.rev.2.bt2 under Drosophila_melanogaster/Ensembl/BDGP5/Sequence/Bowtie2Index/ directory. This takes about 4 minutes 37 seconds on my Core-i7 Laptop computer running xubuntu 12.04.

When I try to run bowtie2-build on mouse genome, it is extremely slow. Unfortunately bowtie2-build does not have "-p" option like bowtie2 to speed it up. 

bowtie2-build -f Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa Dme1_BDGP5_70
brb@brbweb4:~/Anders2013$ ls -lt *.bt2
-rw-r--r-- 1 brb brb 58770281 Feb 10 12:06 Dme1_BDGP5_70.rev.1.bt2
-rw-r--r-- 1 brb brb 40591960 Feb 10 12:06 Dme1_BDGP5_70.rev.2.bt2
-rw-r--r-- 1 brb brb 58770281 Feb 10 12:04 Dme1_BDGP5_70.1.bt2
-rw-r--r-- 1 brb brb 40591960 Feb 10 12:04 Dme1_BDGP5_70.2.bt2
-rw-r--r-- 1 brb brb   339200 Feb 10 12:01 Dme1_BDGP5_70.3.bt2
-rw-r--r-- 1 brb brb 40591953 Feb 10 12:01 Dme1_BDGP5_70.4.bt2

Create a metadata table called "samples"

sri$LibraryName = gsub("S2_DRSC_","",sri$LibraryName) # trim label
samples = unique(sri[,c("LibraryName","LibraryLayout")])
for(i in seq_len(nrow(samples))) {
 rw = (sri$LibraryName==samples$LibraryName[i])
 if(samples$LibraryLayout[i]=="PAIRED") {
   samples$fastq1[i] = paste0(sri$Run[rw],"_1.fastq",collapse=",")
   samples$fastq2[i] = paste0(sri$Run[rw],"_2.fastq",collapse=",")
 } else {
   samples$fastq1[i] = paste0(sri$Run[rw],".fastq",collapse=",")
   samples$fastq2[i] = ""
 }
}

samples$condition = "CTL"
samples$condition[grep("RNAi",samples$LibraryName)] = "KD"
samples$shortname = paste( substr(samples$condition,1,2),
                                         substr(samples$LibraryLayout,1,2), seq_len(nrow(samples)), sep=".")
samples
     LibraryName LibraryLayout
1     Untreated-3        PAIRED
3     Untreated-4        PAIRED
5   CG8144_RNAi-3        PAIRED
7   CG8144_RNAi-4        PAIRED
144   Untreated-1        SINGLE
150 CG8144_RNAi-1        SINGLE
156   Untreated-6        SINGLE
                                                                                            fastq1
1                                                               SRR031714_1.fastq,SRR031715_1.fastq
3                                                               SRR031716_1.fastq,SRR031717_1.fastq
5                                                               SRR031724_1.fastq,SRR031725_1.fastq
7                                                               SRR031726_1.fastq,SRR031727_1.fastq
144 SRR031708.fastq,SRR031709.fastq,SRR031710.fastq,SRR031711.fastq,SRR031712.fastq,SRR031713.fastq
150 SRR031718.fastq,SRR031719.fastq,SRR031720.fastq,SRR031721.fastq,SRR031722.fastq,SRR031723.fastq
156                                                                 SRR031728.fastq,SRR031729.fastq
                                fastq2 condition shortname
1   SRR031714_2.fastq,SRR031715_2.fastq       CTL   CT.PA.1
3   SRR031716_2.fastq,SRR031717_2.fastq       CTL   CT.PA.2
5   SRR031724_2.fastq,SRR031725_2.fastq        KD   KD.PA.3
7   SRR031726_2.fastq,SRR031727_2.fastq        KD   KD.PA.4
144                                           CTL   CT.SI.5
150                                            KD   KD.SI.6
156                                           CTL   CT.SI.7

On the other hand, the <samples.txt> file used in BRB-DGE looks like 

$ cat samples.txt
LibraryName     LibraryLayout   fastq1  fastq2
Untreated-3     PAIRED  SRR031714_1.fastq,SRR031715_1.fastq     SRR031714_2.fastq,SRR031715_2.fastq
Untreated-4     PAIRED  SRR031716_1.fastq,SRR031717_1.fastq     SRR031716_2.fastq,SRR031717_2.fastq
CG8144_RNAi-3   PAIRED  SRR031724_1.fastq,SRR031725_1.fastq     SRR031724_2.fastq,SRR031725_2.fastq
CG8144_RNAi-4   PAIRED  SRR031726_1.fastq,SRR031727_1.fastq     SRR031726_2.fastq,SRR031727_2.fastq
Untreated-1     SINGLE  SRR031708.fastq,SRR031709.fastq,SRR031710.fastq,SRR031711.fastq,SRR031712.fastq,SRR031713.fastq
CG8144_RNAi-1   SINGLE  SRR031718.fastq,SRR031719.fastq,SRR031720.fastq,SRR031721.fastq,SRR031722.fastq,SRR031723.fastq
Untreated-6     SINGLE  SRR031728.fastq,SRR031729.fastq

Align the reads to the reference genome using tophat2 (fastq -> bam, bed)

Note:

  1. '-p' specifies the number of threads to use, -o specifies the output directory. The first name (bowind) is the name of the index (built in advance).
  2. It seems even the program cannot find the reference genome, it still runs.
  3. The run time is about 3 to 4 hours per sample when 7 jobs were running at the same time (5 threads for each sample).
  4. If only one sample was run it took 45 minutes as the paper said (5 threads). If I specify 1 thread, the running time is 1 hour 44 minutes.
  5. If I only run 2 jobs using batch, it takes 1 hour and 1 hour 2 mintues to finish these two jobs.
  6. The input is fastq files. The output is a directory contains 7 files (including *.bam) and 1 subdirectory of logs.
gf = "Drosophila_melanogaster.BDGP5.70.gtf"
bowind = "Dme1_BDGP5_70"  # bt2 files
cmd = with(samples, paste("tophat2 -G", gf, "-p 5 -o", LibraryName,
          bowind, fastq1, fastq2))

cmd # Actually we want to use system() to run each line of cmd.
[1] "tophat2 -G Drosophila_melanogaster.BDGP5.70.gtf -p 5 -o Untreated-3 Dme1_BDGP5_70 SRR031714_1.fastq,SRR031715_1.fastq SRR031714_2.fastq,SRR031715_2.fastq"                           
[2] "tophat2 -G Drosophila_melanogaster.BDGP5.70.gtf -p 5 -o Untreated-4 Dme1_BDGP5_70 SRR031716_1.fastq,SRR031717_1.fastq SRR031716_2.fastq,SRR031717_2.fastq"                           
[3] "tophat2 -G Drosophila_melanogaster.BDGP5.70.gtf -p 5 -o CG8144_RNAi-3 Dme1_BDGP5_70 SRR031724_1.fastq,SRR031725_1.fastq SRR031724_2.fastq,SRR031725_2.fastq"                         
[4] "tophat2 -G Drosophila_melanogaster.BDGP5.70.gtf -p 5 -o CG8144_RNAi-4 Dme1_BDGP5_70 SRR031726_1.fastq,SRR031727_1.fastq SRR031726_2.fastq,SRR031727_2.fastq"                         
[5] "tophat2 -G Drosophila_melanogaster.BDGP5.70.gtf -p 5 -o Untreated-1 Dme1_BDGP5_70 SRR031708.fastq,SRR031709.fastq,SRR031710.fastq,SRR031711.fastq,SRR031712.fastq,SRR031713.fastq "  
[6] "tophat2 -G Drosophila_melanogaster.BDGP5.70.gtf -p 5 -o CG8144_RNAi-1 Dme1_BDGP5_70 SRR031718.fastq,SRR031719.fastq,SRR031720.fastq,SRR031721.fastq,SRR031722.fastq,SRR031723.fastq "
[7] "tophat2 -G Drosophila_melanogaster.BDGP5.70.gtf -p 5 -o Untreated-6 Dme1_BDGP5_70 SRR031728.fastq,SRR031729.fastq "

Tophat options

  • --coverage-search/--no-coverage-search: According to this, it is a step to define possible junctions between exons. If you only want the expression profile of the annotated genes, you can skip this step for speed. The identification of new regions with the coverage is relevant only if you want to detect new splice sites in alternate transcripts or even new genes.
  • -r/--mate-inner-dist <int>: This is the expected (mean) inner distance between mate pairs. For, example, for paired end runs with fragments selected at 300bp, where each end is 50bp, you should set -r to be 200. The default is 50bp. Check out this post about fragment size & inner size. This post discusses about the fragment size.
  • -g/--max-multihits <int>: Instructs TopHat to allow up to this many alignments to the reference for a given read, and choose the alignments based on their alignment scores if there are more than this number. The default is 20 for read mapping.
  • --library-type: The default is unstranded (fr-unstranded). If either fr-firststrand or fr-secondstrand is specified, every read alignment will have an XS attribute tag.
  • --GTF option: Tophat now takes the approach of mapping the reads on the transcriptome first, with only the unmapped reads being further aligned to the whole genome and going through the novel junction discovery process like before. Please note that the values in the first column of the provided GTF/GFF file (column which indicates the chromosome or contig on which the feature is located), must match the name of the reference sequence in the Bowtie index you are using with TopHat.
  • -T/--transcriptome-only: only align the reads to the transcriptome and report only those mappings as genomic mappings. Note that we still get junctions map.
  • -x/--transcriptome-max-hits: Maximum number of mappings allowed for a read, when aligned to the transcriptome (any reads found with more then this number of mappings will be discarded).

Some helpful information:

Output files

$ ls -lh Untreated-6
total 2.2G
-rw-rw-r-- 1 brb brb 2.0G May  6 18:11 accepted_hits.bam
-rw-rw-r-- 1 brb brb  202 May  6 18:03 align_summary.txt
-rw-rw-r-- 1 brb brb 2.0M May  6 18:03 deletions.bed
-rw-rw-r-- 1 brb brb 612K May  6 18:03 insertions.bed
-rw-rw-r-- 1 brb brb 2.7M May  6 18:03 junctions.bed
drwxrwxr-x 2 brb brb 4.0K May  6 18:11 logs
-rw-rw-r-- 1 brb brb   70 May  6 17:43 prep_reads.info
-rw-rw-r-- 1 brb brb 197M May  6 18:12 unmapped.bam

$ ls -lh Untreated-6/logs
total 108K
-rw-rw-r-- 1 brb brb    0 May  6 18:11 bam_merge_um.log
-rw-rw-r-- 1 brb brb  12K May  6 17:59 bowtie_build.log
-rw-rw-r-- 1 brb brb    0 May  6 17:39 bowtie_inspect_recons.log
-rw-rw-r-- 1 brb brb  221 May  6 17:53 bowtie.left_kept_reads.log
-rw-rw-r-- 1 brb brb  216 May  6 17:59 bowtie.left_kept_reads_seg1.log
-rw-rw-r-- 1 brb brb  216 May  6 17:59 bowtie.left_kept_reads_seg2.log
-rw-rw-r-- 1 brb brb  215 May  6 18:00 bowtie.left_kept_reads_seg3.log
-rw-rw-r-- 1 brb brb  394 May  6 17:59 juncs_db.log
-rw-rw-r-- 1 brb brb  300 May  6 18:00 long_spanning_reads.segs.log
-rw-rw-r-- 1 brb brb  105 May  6 17:43 prep_reads.log
-rw-rw-r-- 1 brb brb  617 May  6 18:03 reports.log
-rw-rw-r-- 1 brb brb    0 May  6 18:07 reports.merge_bam.log
-rw-rw-r-- 1 brb brb   40 May  6 18:05 reports.samtools_sort.log0
-rw-rw-r-- 1 brb brb   40 May  6 18:05 reports.samtools_sort.log1
-rw-rw-r-- 1 brb brb   40 May  6 18:04 reports.samtools_sort.log10
-rw-rw-r-- 1 brb brb   40 May  6 18:05 reports.samtools_sort.log2
-rw-rw-r-- 1 brb brb   40 May  6 18:04 reports.samtools_sort.log3
-rw-rw-r-- 1 brb brb   40 May  6 18:06 reports.samtools_sort.log4
-rw-rw-r-- 1 brb brb   40 May  6 18:06 reports.samtools_sort.log5
-rw-rw-r-- 1 brb brb   40 May  6 18:05 reports.samtools_sort.log6
-rw-rw-r-- 1 brb brb   40 May  6 18:05 reports.samtools_sort.log7
-rw-rw-r-- 1 brb brb   40 May  6 18:04 reports.samtools_sort.log8
-rw-rw-r-- 1 brb brb   40 May  6 18:04 reports.samtools_sort.log9
-rw-rw-r-- 1 brb brb  12K May  6 18:12 run.log
-rw-rw-r-- 1 brb brb  813 May  6 17:58 segment_juncs.log
-rw-rw-r-- 1 brb brb 2.0K May  6 18:12 tophat.log

transcriptome_data

If we specify --transcriptome-index=transcriptome_data/known in tophat2 command, we shall get a directory transcriptome with several files 

known.1.bt2
known.2.bt2
known.3.bt2
known.4.bt2
known.ver
known.fa
known.fa.tlst
known.gff

Output from console

[2014-04-18 16:42:12] Beginning TopHat run (v2.0.10)
-----------------------------------------------
[2014-04-18 16:42:12] Checking for Bowtie
		  Bowtie version:	 2.1.0.0
[2014-04-18 16:42:12] Checking for Samtools
		Samtools version:	 0.1.19.0
[2014-04-18 16:42:12] Checking for Bowtie index files (genome)..
[2014-04-18 16:42:12] Checking for reference FASTA file
[2014-04-18 16:42:12] Generating SAM header for genome
[2014-04-18 16:42:58] Reading known junctions from GTF file
[2014-04-18 16:43:17] Preparing reads
	 left reads: min. length=49, max. length=49, 26721504 kept reads (2587 discarded)
[2014-04-18 16:47:40] Building transcriptome data files E2_Rep2/tmp/genes
[2014-04-18 16:48:22] Building Bowtie index from genes.fa
[2014-04-18 17:12:33] Mapping left_kept_reads to transcriptome genes with Bowtie2 
[2014-04-18 17:44:09] Resuming TopHat pipeline with unmapped reads
[2014-04-18 17:44:09] Mapping left_kept_reads.m2g_um to genome genome with Bowtie2 
[2014-04-18 17:49:50] Mapping left_kept_reads.m2g_um_seg1 to genome genome with Bowtie2 (1/2)
[2014-04-18 17:51:07] Mapping left_kept_reads.m2g_um_seg2 to genome genome with Bowtie2 (2/2)
[2014-04-18 17:51:54] Searching for junctions via segment mapping
	Coverage-search algorithm is turned on, making this step very slow
	Please try running TopHat again with the option (--no-coverage-search) if this step takes too much time or memory.
[2014-04-18 18:08:13] Retrieving sequences for splices
[2014-04-18 18:10:10] Indexing splices
[2014-04-18 18:19:15] Mapping left_kept_reads.m2g_um_seg1 to genome segment_juncs with Bowtie2 (1/2)
[2014-04-18 18:21:26] Mapping left_kept_reads.m2g_um_seg2 to genome segment_juncs with Bowtie2 (2/2)
[2014-04-18 18:23:38] Joining segment hits
[2014-04-18 18:25:52] Reporting output tracks
-----------------------------------------------
[2014-04-18 18:38:05] A summary of the alignment counts can be found in E2_Rep2/align_summary.txt
[2014-04-18 18:38:05] Run complete: 01:55:52 elapsed

Some lessons

The following table summarizes the result of using '-G' and '--transcriptome-index' options. In conclusion, 3 possible ways of using these 2 options:

  1. Not using '-G'
  2. Using '-G' but no '--transcriptome-index'
  3. Run tophat2 one time to generate transcriptome index files and then run tophat2 again with '-G' and '--transcriptome-index' options.
Example 1 (fail) Wrong spec --transcriptome-index
Example 2 (fail) Using -G & --transcriptome-index but my files (genes.gtf & genome.*)
Example 3 (OK) No -G option
Example 4 (OK) Proper use of using -G & --transcriptome-index (2 steps)
Example 5 (fail) Using -G & --transcriptome-index using Illumina iGenomes dir
Example 6 (fail) Like Example 5 but cp genes.gtf to genome.gtf
Example 7 (OK) Using -G but no --transcriptome-index

More details can be found on my github website.

To create the transcriptome index folder, it is not necessary to allocate too much memory or multithreads. The 'swarm' and 'jobload' commands show 0.3 GB memory and 1 thread is used even we allocate more memory and more CPUs. It took about 15 minutes to create the folder when the following code is used 

tophat2 -p 2 \
    -G "/fdb/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf" \
    --transcriptome-index=transcriptome_data/known \
    "/fdb/igenomes/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome"

This post discussed Tophat with and without GTF.

FAQs for Tophat

  • TopHat2 on multiple samples, avoid building Bowtie index from genes.fa each time?

Is it necessary to run the following code by Tophat? See this post on seqanswers.com. 

[2014-09-18 10:38:45] Building transcriptome data files /tmp/genes
[2014-09-18 10:39:21] Building Bowtie index from genes.fa

For other users, which may encounter the same challenge - The trick is to run this command first: 

tophat2 -G iGenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf \
       --transcriptome-index=transcriptome_data/known \
       iGenomes/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome

and then subsequently call tophat2 with this command: 

tophat2 --num-threads 12 --transcriptome-index=transcriptome_data/known \
        iGenomes/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome 
        myfastq_R1.fastq.gz myfastq_R2.fastq.gz

After running the above command, you'll see the following 1 line (instead of 2 lines as shown above) 

[2014-09-18 12:12:04] Using pre-built transcriptome data..

Which is significantly faster, when running multiple samples.

The UCSC/hg19 data can retrieved like so: 

wget ftp://igenome:[email protected]/Homo_sapiens/UCSC/hg19/Homo_sapiens_UCSC_hg19.tar.gz

Organize, sort and index the BAM files and create SAM files (bam -> sam)

Note

  1. Potentially we can run the commands in parallel. This step needs lots of disk I/O so it may be better not to run the command in parallel.
  2. Four steps:
    1. first step - Input: original aligned bam file. Output: <Untreated-3_sn.bam> (1.5 GB). sort alignment file by read name (-n option).
    2. Second step - Input: sorted bam file from 1st step. Output: <Untreated-3_sn.sam> (11 GB). BAM<->SAM conversion. The SAM file generated from these 2 steps will be used in HTSEQ-count (For paired-end data, the alignment have to be sorted either by read name or by alignment position; see htseq-count documentation).
    3. Third step - Input: original aligned bam file. Output: <Untreated-3_s.bam> (1.2 GB). sort alignment file by chromosomal coordinates.
    4. Fourth step - Input: sorted bam file from 3rd step. Output: <Untreated-3_s.bam.bai> (375 KB). index alignment. The SAM and BAI files generated from these 2 steps together with GTF file will be used in IGV (IGV requires that both SAM and BAM files be sorted by position and indexed; see IGV documentation).
  3. Examples how to use. See here

If we use samtools view to see the sorted bam files, we may find some fagments (first column) has only one read in this file and some fragments are not in this file.

If we look into the (sorted by name) unmapped.bam file, we can see the unmapped fragments. 

for(i in seq_len(nrow(samples))) {
 lib = samples$LibraryName[i]
 ob = file.path(lib, "accepted_hits.bam")
 # sort by name, convert to SAM for htseq-count
 cat(paste0("samtools sort -n ",ob," ",lib,"_sn"),"\n")
 cat(paste0("samtools view -o ",lib,"_sn.sam ",lib,"_sn.bam"),"\n")
 # sort by position and index for IGV
 cat(paste0("samtools sort ",ob," ",lib,"_s"),"\n")
 cat(paste0("samtools index ",lib,"_s.bam"),"\n\n")
}

samtools sort -n Untreated-3/accepted_hits.bam Untreated-3_sn
samtools view -o Untreated-3_sn.sam Untreated-3_sn.bam
samtools sort Untreated-3/accepted_hits.bam Untreated-3_s
samtools index Untreated-3_s.bam

samtools sort -n Untreated-4/accepted_hits.bam Untreated-4_sn
samtools view -o Untreated-4_sn.sam Untreated-4_sn.bam
samtools sort Untreated-4/accepted_hits.bam Untreated-4_s
samtools index Untreated-4_s.bam

samtools sort -n CG8144_RNAi-3/accepted_hits.bam CG8144_RNAi-3_sn
samtools view -o CG8144_RNAi-3_sn.sam CG8144_RNAi-3_sn.bam
samtools sort CG8144_RNAi-3/accepted_hits.bam CG8144_RNAi-3_s
samtools index CG8144_RNAi-3_s.bam 

samtools sort -n CG8144_RNAi-4/accepted_hits.bam CG8144_RNAi-4_sn
samtools view -o CG8144_RNAi-4_sn.sam CG8144_RNAi-4_sn.bam
samtools sort CG8144_RNAi-4/accepted_hits.bam CG8144_RNAi-4_s
samtools index CG8144_RNAi-4_s.bam

samtools sort -n Untreated-1/accepted_hits.bam Untreated-1_sn
samtools view -o Untreated-1_sn.sam Untreated-1_sn.bam
samtools sort Untreated-1/accepted_hits.bam Untreated-1_s
samtools index Untreated-1_s.bam

samtools sort -n CG8144_RNAi-1/accepted_hits.bam CG8144_RNAi-1_sn
samtools view -o CG8144_RNAi-1_sn.sam CG8144_RNAi-1_sn.bam
samtools sort CG8144_RNAi-1/accepted_hits.bam CG8144_RNAi-1_s
samtools index CG8144_RNAi-1_s.bam

samtools sort -n Untreated-6/accepted_hits.bam Untreated-6_sn
samtools view -o Untreated-6_sn.sam Untreated-6_sn.bam
samtools sort Untreated-6/accepted_hits.bam Untreated-6_s
samtools index Untreated-6_s.bam

Output from console

[bam_sort_core] merging from 15 files...

Check Coverage from bam file

Use samtools mpileup or Qualimap software as commented inbiostars.

The output of samtools mpileup has a pileup format. For example, in the GSE37918 dataset, 

$ wc -l MDA-MB-231-control_mpileup.txt

$ head -n 2 MDA-MB-231-control_mpileup.txt
1  12060  N  1  ^!C  B
1  12061  N  1  T    C

where each line consists of chromosome, 1-based coordinate, reference base, the number of reads covering the site, read bases and base qualities.

Or use igvtools https://hpc.nih.gov/apps/IGV.html

Inspect alignments with IGV

The Integrative Genomics Viewer (IGV) is a high-performance visualization tool for interactive exploration of large, integrated genomic datasets. It supports a wide variety of data types, including array-based and next-generation sequence data, and genomic annotations.

Launch

I can launch IGV in a terminal (suppose the file IGV_2.3.52.zip is extracted to the directory called binary under the HOME directory): 

 ~/binary/IGV_2.3.52/igv.sh

Note that in addition to the bam file, we need to run samtools index XXX.bam to create the bai file before IGV can use it.

Command line options

bash igv.sh input.bam -g hg19 -b igv_batch_script.txt

And a sample <igv_batch_script.txt> file 

new
genome hg19
load input.bam
snapshotDirectory /path/to/igv_screenshots
goto chr19:59418052-59418053
sort base
collapse
snapshot chr19_59418052-59418053.png
goto chr1:15680529-15680532 chr3:5680521-5680533 
sort base
collapse
snapshot chr1_15680529-15680532.chr3_5680521-5680533.png
exit

Genomes

IGV contains a lot of genomes. We can access them by using the drop-down list box and select 'More ...' entry. See Hosted Genomes for more information about the meaning of this list (some of them on the list do not appear on the website though).

To create .genome file, following the steps:

  • Download reference genome fasta file (bt2 won't work)
wget ftp://ftp.ensembl.org/pub/release-70/fasta/drosophila_melanogaster/dna/Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa.gz
gunzip Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa.gz
  • Make sure gene model annotation file is available (*.gtf)
  • If you have the cytoband file, IGV can display the chromosome ideogram. Otherwise, we won't see the ideogram. Unfortunately I don't see a download choice for the cytoband on the ensembl.org website. The UCSC website does provide the cytoband.txt file for downloading.
  • In IGV, click Genomes > Create .genom File. Enter 'Anders2013' for both Unique identifer & Descriptive name. Browse the right FASTA file and Gene file. Click OK. We can save the file <Anders2013.genome> under the data directory.

If the above steps work, we should be able to prepare a sorted bam file with its index file to be used in IGV. For example, 

samtools sort Untreated-6/accepted_hits.bam Untreated-6_s
samtools index Untreated-6_s.bam

Splice junctions

The junctions track calls a splicing event when at least a single read splits across two exons in the alignment track.

IGV defines exons by your sample's read alignments.

Each splice junction is represented by an arc from the beginning to the end of the junction.

  • Junctions from the + strand are colored red and extend above the center line.
  • Junctions from the – strand are blue and extend below the center line.

See an example of it in IGV from GTF.

The bed file format is specified in genome.ucsc.edu and a few lines of <junctions.bed> are given below. 

brb@brb-T3500:~/Anders2013/Untreated-6$ head junctions.bed 
track name=junctions description="TopHat junctions"
2L	11095	11479	JUNC00000001	4	-	11095	11479	255,0,0	2	74,70	0,314
2L	11271	11479	JUNC00000002	24	-	11271	11479	255,0,0	2	73,70	0,138
2L	11447	11842	JUNC00000003	45	-	11447	11842	255,0,0	2	71,64	0,331

By default IGV won't show the splice junction. Before loading data, check Show junction track in the Alignment Preferences panel. The panel's settings must be adjusted for your data as default settings are for genomic reads for which splicing is irrelevant.

Below is an example from BRB-SeqTools GSE11209 dataset (Saccharomyces_cerevisiae 酵母屬 yeast).

Igv gse11209 junction.png 

brb@T3600 ~/Downloads/testdata/GSE11209/dT_ori $ cat junctions.bed 
track name=junctions description="TopHat junctions"
I	142233	142632	JUNC00000001	3	+	142233	142632	255,0,0	2	20,13	0,386
II	60161	60721	JUNC00000002	6	-	60161	60721	255,0,0	2	32,24	0,536
II	332859	333403	JUNC00000003	1	+	332859	333403	255,0,0	2	16,17	0,527
...

Small test data from Tophat (paired end)

The following screenshot was taken from another (small) dataset with two fq files (paired end). There are 100 sequences in the fq file. The reference genome has only 13 sequences. See also this post.

Before using IGV, we need to run Tophat and samtools (one is to get sorted by position in the reference in bam format, and the other is to get an index file in bai format). Both bam and bai files are binary. If we just use the bam file generated from Tophat in IGV, IGV will show a message 'An index file is required for SAM & BAM files'. See the BAM file requirement in IGV documentation. 

wget http://ccb.jhu.edu/software/tophat/downloads/test_data.tar.gz
tar -xzf test_data.tar.gz

# Assume required software are installed; e.g. through BDGE.
export bdge_bowtie_PATH=/opt/RNA-Seq/bin/bowtie2-2.2.1
export bdge_tophat_PATH=/opt/RNA-Seq/bin/tophat-2.0.11.Linux_x86_64
export bdge_samtools_PATH=/opt/RNA-Seq/bin/samtools-0.1.19:/opt/RNA-Seq/bin/samtools-0.1.19/bcftools:/opt/RNA-Seq/bin/samtools-0.1.19/misc
export PATH=$bdge_bowtie_PATH:$bdge_samtools_PATH:$bdge_tophat_PATH:$PATH

tophat -r 20 test_ref reads_1.fq reads_2.fq 
# sort by location and index
samtools sort tophat_out/accepted_hits.bam -o tophat_out_s.bam -@ 11  # Output tophat_out_s.bam
samtools index tophat_out_s.bam                                       # Output tophat_out_s.bam.bai

The "-r" option in tophat is related to mate-inner-dist. See the discussion here.

IGV.png IGV whole.png IGV whole color.png

  1. Genome -> Load genome from file -> test_ref.fa
  2. File -> load from file -> tophat_out_s.bam (read_1.fq won't work)
  3. We can change the color of alignment by read strand by using the right click menu on *.bam window.

When we launch IGV, it is better to increase the default memory size (2GB). To do that, open igv.sh file and change -Xmx2000m to -Xmx4000m, for example to increase the memory from 2GB to 4GB.

  • The reference genome (*.fa file) is shown on the bottom of IGV.
  • Mouse over a sequence will show "Read name" (we can go back to fastq file to double check the sequence), "Location" (the location from mouse over), "Alignment start" (the start position).
  • If there is a mismatch from a bp, it will be shown in a different color (T-red, C-blue, A-green, G-brown).
  • The histogram-like plot is called Coverage.
  • One sequence may be splitted after alignment.
  • The <align_summary.txt> file (see below for its content) shows the number of reads in input and number of mapped reads with percentage of mapped.
  • There are 142 reads shown on the IGV (verified by <align_summary.txt>).

The <test_ref.fa> file (reference genome) is shown below. The total length is 50 * 13 = 650 bp. We can verify this by looking at both the reference genome (bottom) and bp scale (top). 

>test_chromosome
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
ACTACTATCTGACTAGACTGGAGGCGCTTGCGACTGAGCTAGGACGTGCC
ACTACGGGGATGACGACTAGGACTACGGACGGACTTAGAGCGTCAGATGC
AGCGACTGGACTATTTAGGACGATCGGACTGAGGAGGGCAGTAGGACGCT
ACGTATTTGGCGCGCGGCGCTACGGCTGAGCGTCGAGCTTGCGATACGCC
GTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAG
ACTATTACTTTATTATCTTACTCGGACGTAGACGGATCGGCAACGGGACT
GTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAG
TTTTCTACTTGAGACTGGGATCGAGGCGGACTTTTTAGGACGGGACTTGC
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

The <align_summary.txt> file is 

Left reads:
          Input     :       100
           Mapped   :        72 (72.0% of input)
Right reads:
          Input     :       100
           Mapped   :        70 (70.0% of input)
71.0% overall read mapping rate.

Aligned pairs:        50
50.0% concordant pair alignment rate.

The first 2 reads (read length is 75) in <reads_1.fq> file looks like 

@test_mRNA_150_290_0/1
TCCTAAAAAGTCCGCCTCGGTCTCAGTCTCAAGTAGAAAAAGTCCCGTTGGCGATCCGTCTACGTCCGAGTAAGA
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@test_mRNA_8_197_1/1
TCTGACTAGACTGGAGGCGCTTGCGACTGAGCTAGGACGTGACACTACGGGGATGGCGACTAGGACTACGGACGG
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

The first 2 reads in <reads_2.fq> file looks like 

@test_mRNA_150_290_0/2
TACGTATTTGTCGCGCGGCCCTACGGCTGAGCGTCGAGCTTGCGATCCGCCACTATTACTTTATTATCTTACTCG
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@test_mRNA_8_197_1/2
GTATCGCAAGCTCGACGCTCAGCCGTAGGGCCGCGCGCCAAATACGTAGCGTCCTACTGCCCTCCTCAGTCCGAT
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

The IGV guide here shows how to interpret the colors for alignment data. For example, read bases that match are displayed in gray.

IGV Channel is a youtube channel where it contains some videos about the IGV.

Paire end sequencing

For paired end data, it is useful to use the right-click menu and select

  1. 'View as pairs' option. Pairs will be joined by a line.
  2. Color alignments by read strand.

The alignment of the right read works by 1) reverse the order and 2) apply compliment operator. For example, the following shows the reference sequence on the location of @test_mRNA_6_182_59/2. As we can see if we flip & apply the compliment of the sequence of @test_mRNA_6_182_59/2, they are almost matched except one base pair. 

Reference
GGACTATTTAGGACGATCGGACTGAGGAGGGCAGTAGGACGCTACGTATTTGGCGCGCGGCGCTACGGCTGAGCG

@test_mRNA_6_182_59/2
CGCTCAGCCGTAGGGCCGCGCGCCAAATACGTAGCGTCCTACTGCCCTCCTCAGTCCGATCGTCCTAAATAGTCC

SRA000299 (single end)

This data was used in this post.

There are 4 libraries and 6 fastq files. The samples.txt is given below. 

LibraryName     LibraryLayout   fastq1  fastq2
Kidney_0007_1.5pM       SINGLE  SRR002324.fastq
Kidney_0007_3pM SINGLE  SRR002320.fastq,SRR002325.fastq
Liver_0007_1.5pM        SINGLE  SRR002322.fastq
Liver_0007_3pM  SINGLE  SRR002321.fastq,SRR002323.fastq

Anders2013 (mix of single & paired end)

This is the data used in this wiki page. The sample meta data can be found at here.

Here we focus on a single-end library Untreated-6 which contains 2 fastq files (SRR031728.fastq, SRR031729.fastq).

  • In IGV, select the chromosome 2RHet and zoom in to the region of 1,638,560 bp. We are using the ref genome from ensembl.org as Anders2013 paper has used. Note the bp location number always starts at 1 for each chromosome. Here I am focusing on the read SRR031729.8202948 which has a color of a negative strand even the data is single end.
# Find the read sequence. First we find the line number using '-n' option of the 'grep' command
brb@brb-T3500:~/Anders2013$ grep -n 8202948 SRR031729.fastq        
32811789:@SRR031729.8202948 HWI-EAS299:3:65:192:596 length=75
32811791:+SRR031729.8202948 HWI-EAS299:3:65:192:596 length=75

# Next we display the text from line 32811789 to 32811790
brb@brb-T3500:~/Anders2013$ sed -n '32811789,32811790p'  SRR031729.fastq
@SRR031729.8202948 HWI-EAS299:3:65:192:596 length=75
CTTATCCTTTCTCTCTTGTATTTCCTGTGGAGGAAATTGACCTCAACCCATGGACTACCGAAACCTGGCAATATC
  • Using an online DNA tool we can get the complement and inverse of the sequence (we only need to do this extra step for a negative strand.
GATATTGCCAGGTTTCGGTAGTCCATGGGTTGAGGTCAATTTCCTCCACAGGAAATACAAGAGAGAAAGGATAAG
  • The reference genome shows the read starts at 2RHet:1638559, Cigar 75M. I write down the sequence by selecting 'Show all bases' in right-click menu. Not that the alignment is 100% for this read.
GATATTGCCAGGTTTCGGTAGTCCATGGGTTGAGGTCAATTTCCTCCACAGGAAATACAAGAGAGAAAGGATAAG

The ref genome seq matched with the complement & reverse seq of the (negative strand; shown in a purple color) read. This verifies the plot.

IGB

It is also a java-based software. Java version 1.8 is required.

  • It provides similar functions as IGV.
  • The memory handling is not as good as IGV (tested using Anders2013 data with the same 4000M setting).
  • There are several tutorial videos for IGB and the user guide is good.

Other visualization tools

CHAPTER V : Visualizing next generation sequencing data

Count reads using htseq-count (sam -> count)

samples$countf = paste(samples$LibraryName, "count", sep=".")
gf = "Drosophila_melanogaster.BDGP5.70.gtf"
cmd = paste0("htseq-count -s no -a 10 ",
            samples$LibraryName, "_sn.sam ",
            gf," > ", samples$countf)

cmd
[1] "htseq-count -s no -a 10 Untreated-3_sn.sam Drosophila_melanogaster.BDGP5.70.gtf > Untreated-3.count"    
[2] "htseq-count -s no -a 10 Untreated-4_sn.sam Drosophila_melanogaster.BDGP5.70.gtf > Untreated-4.count"    
[3] "htseq-count -s no -a 10 CG8144_RNAi-3_sn.sam Drosophila_melanogaster.BDGP5.70.gtf > CG8144_RNAi-3.count"
[4] "htseq-count -s no -a 10 CG8144_RNAi-4_sn.sam Drosophila_melanogaster.BDGP5.70.gtf > CG8144_RNAi-4.count"
[5] "htseq-count -s no -a 10 Untreated-1_sn.sam Drosophila_melanogaster.BDGP5.70.gtf > Untreated-1.count"    
[6] "htseq-count -s no -a 10 CG8144_RNAi-1_sn.sam Drosophila_melanogaster.BDGP5.70.gtf > CG8144_RNAi-1.count"
[7] "htseq-count -s no -a 10 Untreated-6_sn.sam Drosophila_melanogaster.BDGP5.70.gtf > Untreated-6.count"

Output from console

Lots of output 

....
37200000 SAM alignment records processed.
37300000 SAM alignment records processed.
37399340 SAM alignments  processed.

-r pos option and 'Maximum alignment buffer size exceeded error'

If the input bam file is sorted by position, we can use -r pos option in HTSeq.scripts.count program. However, I got an error complaining Maximum alignment buffer size exceeded while pairing SAM alignments. Following the author's suggestion on the post, it is better to sort the bam file by read name and skip the -r option.

Performance

http://seqanswers.com/forums/showthread.php?t=18920 htseq-count was designed to use very little memory] (0.1GB from my observation). But the price is it is single thread and may takes several several hours. Note: Another tool is CuffLinks. See Sean Davis's tutorial.

Error: 'pair_alignments' needs a sequence of paired-end alignments

http://seqanswers.com/forums/showthread.php?t=41785 & https://www.biostars.org/p/111221/

Run the following to check if there are single-end in the bam files 

samtools view -cF 1 bamfile

It it has, run the following to separate single-end and paired-end reads 

samtools view -bf 1 foo.bam > foo.paired-end.bam
samtools view -bF 1 foo.bam > foo.single-end.bam

Then run htseq-count separately and then combine the counts.

summarizeOverlaps() from GenomicAlignments package

http://www.bioconductor.org/packages/release/bioc/vignettes/GenomicAlignments/inst/doc/summarizeOverlaps.pdf

Note that GenomicAlignments can read BAM files.

ReadCounter - htseq alternative

  • it's multi threaded
  • can produce the same result as htseq
  • java based

featureCount from Subread

  • Subread: high-performance read alignment, quantification and mutation discovery
  • featureCounts: a ultrafast and accurate read summarization program

DESeq2 normalization

install.packages("~/Downloads/DESeq2paper_1.3.tar.gz", repos = NULL, type="source")
source("http://bioconductor.org/biocLite.R") # https:// not work on my R 3.2.5 Ubuntu
biocLite("DESeq2", ask=FALSE)
# Got the following error because RcppArmidillo requires the following two libraries
# /usr/bin/ld: cannot find -llapack
# /usr/bin/ld: cannot find -lblas
# Go to ubuntu software center search liblapack and libblas
# select the following 6 components
#   libblas3gf, libblas-doc, libblas-dev, liblapack3gf, liblapack-doc, liblapack-dev
# Then double check ls -l /usr/lib/liblapack*
#                   ls -l /usr/lib/libblas*
biocLite("GenomicRanges", ask=FALSE)
biocLite("GenomicAlignments", ask=FALSE)
install.packages("caret", repos="https://cran.rstudio.com")
  • The count matrix and metadata, including the gene model and sample information, are stored in an S4 class derived from the SummarizedExperiment class of the GenomicRanges package. SummarizedExperiment objects containing count matrices can be easily generated using the summarizeOverlaps() function of the GenomicAlignments package.
  • Log2 fold-change & DESeq2 model in a nutshell (youtube)

Input

DESeqDataSetFromHTSeqCount(sampleTable, directory, design)
# sampleTable is like colData in the next 2 functions

DESeqDataSetFromTximport(txi, colData, design)
# txi is obtained by tximport()

DESeqDataSetFromMatrix(countData, colData, design)
# count data input

DESeqDataSet(se, design) 
# e.g. the ''SummarizedExperiment'' object created by tximeta()
# 'airway' package/object is an example

collapseReplicates

dds <- makeExampleDESeqDataSet(m=12)

# make data with two technical replicates for three samples
dds$sample <- factor(sample(paste0("sample",rep(1:9, c(2,1,1,2,1,1,2,1,1)))))
dds$run <- paste0("run",1:12)

ddsColl <- collapseReplicates(dds, dds$sample, dds$run)
# dds$sample is a grouping variable

# examine the colData and column names of the collapsed data
colData(ddsColl)
colnames(ddsColl)
# DataFrame with 9 rows and 4 columns
#         condition   sample         run runsCollapsed
#          <factor> <factor> <character>   <character>
# sample1         A  sample1        run6     run6,run9
# sample2         B  sample2       run12         run12
# sample3         A  sample3        run2          run2
# sample4         A  sample4        run5     run5,run7
# sample5         A  sample5        run1          run1
# sample6         A  sample6        run4          run4
# sample7         B  sample7        run8    run8,run10
# sample8         A  sample8        run3          run3
# sample9         B  sample9       run11         run11

# check that the sum of the counts for "sample1" is the same
# as the counts in the "sample1" column in ddsColl
matchFirstLevel <- dds$sample == levels(dds$sample)[1]
stopifnot(all(rowSums(counts(dds[,matchFirstLevel])) == counts(ddsColl[,1])))

Normalized counts = raw counts/sizeFactor

  • How to access the normalized data of a DESeqDataSet
  • How exactly DESeq2 correct for differences in sequencing depth and library composition to get normalized counts?
    • To normalize for sequencing depth and RNA composition, DESeq2 uses the median of ratios method. On the user-end there is only one step, but on the back-end there are multiple steps involved. The algorithm performs multiple steps such as computing the pseudo-reference: geometric mean for each gene across all samples.
  • StatQuest: DESeq2 part 1: Library Normalization (480p only)
    1. take the log of all the values (log0 = -Inf)
    2. average each row
    3. filter out genes with infinity
    4. subtract the average log value from the log(counts)
    5. calculate the median of the ratios for each sample
    6. convert the medians to "normal numbers" to get the final scaling factors for each sample (exp(these medians))
    7. divide the original read counts by the scaling factors
  • Summary
    1. Calculate the normalization factor (size factor) for each sample:
      1. Create a pseudo-reference sample: For each gene, calculate the geometric mean of counts across all samples. This geometric mean serves as a pseudo-reference sample.
      2. Calculate the ratio of each sample to the reference: For each gene in each sample, divide the count by the geometric mean (pseudo-reference) calculated in step 1. This gives the ratio of each sample to the reference.
      3. For each sample, calculate the median of the ratios obtained in step 2. This median value is the size factor for that sample.
    2. Calculate the normalized count values: Divide the raw counts for each gene in each sample by the size factor for that sample. This gives the normalized count values.
  • Differential expression analysis for sequence count data for DESeq package from Anders & Huber, 2010. If we want normalized counts, we need to run 1) estimateSizeFactors() for the normalization, and 2) counts(dds, normalized = TRUE) to extract normalized counts or normTransform(dds) on log2 scale. The normalization is part of the DESeq() function so you can extract counts after running that as well. 
    source("http://bioconductor.org/biocLite.R"); biocLite("pasilla")
directory <- system.file("extdata", package="pasilla", mustWork=TRUE)
sampleFiles <- grep("treated",list.files(directory),value=TRUE)
sampleCondition <- sub("(.*treated).*","\\1",sampleFiles)
sampleTable <- data.frame(sampleName = sampleFiles,
                           fileName = sampleFiles,
                           condition = sampleCondition)
ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable,
                                        directory = directory,
                                        design= ~ condition)
dds <- ddsHTSeq[ rowSums(counts(ddsHTSeq)) > 1, ]
dds <- estimateSizeFactors(dds)
filteredlog = counts(dds, normalized=T)
filteredlog0 = counts(dds, normalized=F)

filteredlog0[1:5, 1:5]
#                 treated1fb.txt treated2fb.txt treated3fb.txt untreated1fb.txt untreated2fb.txt
# FBgn0000008:003              0              1              0                1                1
# FBgn0000008:004              1              0              1                0                1
# FBgn0000008:005              4              1              1                2                2
# FBgn0000008:007             18              8              7                8               11
# FBgn0000008:008              4              1              0                0                1

scale(filteredlog0[1:5, ], center=F, scale=sizeFactors(dds))[, 1:5]
#                 treated1fb.txt treated2fb.txt treated3fb.txt untreated1fb.txt untreated2fb.txt
# FBgn0000008:003      0.0000000       1.246732       0.000000        0.9712273        0.6178182
# FBgn0000008:004      0.6815046       0.000000       1.093313        0.0000000        0.6178182
# FBgn0000008:005      2.7260184       1.246732       1.093313        1.9424546        1.2356364
# FBgn0000008:007     12.2670828       9.973855       7.653188        7.7698183        6.7960001
# FBgn0000008:008      2.7260184       1.246732       0.000000        0.0000000        0.6178182

filteredlog[1:5, 1:5]
#                 treated1fb.txt treated2fb.txt treated3fb.txt untreated1fb.txt untreated2fb.txt
# FBgn0000008:003      0.0000000       1.246732       0.000000        0.9712273        0.6178182
# FBgn0000008:004      0.6815046       0.000000       1.093313        0.0000000        0.6178182
# FBgn0000008:005      2.7260184       1.246732       1.093313        1.9424546        1.2356364
# FBgn0000008:007     12.2670828       9.973855       7.653188        7.7698183        6.7960001
# FBgn0000008:008      2.7260184       1.246732       0.000000        0.0000000        0.6178182
    # Retrieve raw counts and size factors
raw_counts <- counts(dds)
size_factors <- sizeFactors(dds)

# Manually calculate normalized counts
manual_normalized_counts <- sweep(raw_counts, 2, size_factors, FUN = "/")

# Retrieve normalized counts from DESeq2
normalized_counts <- counts(dds, normalized = TRUE)

# Compare the two matrices
all.equal(manual_normalized_counts, normalized_counts)

High-Throughput Count Data

http://web.stanford.edu/class/bios221/book/Chap-CountData.html

estimateSizeFactorsForMatrix()

We can compare the output of estimateSizeFactorsForMatrix() with what you get by simply taking the column sums. See High-Throughput Count Data.

Use a robust regression to estimate the size factor instead of taking the sum of reads per sample.

The output estimateSizeFactorsForMatrix() is a vector of the number of samples.

estimateDispersions()

NB dispersion in DESeq2 correspond to Gamma-Poisson parametrisation? 

dds <- makeExampleDESeqDataSet() # 1000 x 12
dds <- estimateSizeFactors(dds)
dds <- estimateDispersions(dds)
length(dispersions(dds))  # [1] 1000

plotDispEsts(dds) # x=mean of normalized counts, y=dispersion
# plots the per-gene dispersion estimates together with 
# the fitted mean-dispersion relationship

So the output of estimateDispersions() is a vector of the number of genes.

DESeq() and gene filtering

Combine two DESeq2 objects (dds) for analysis

How to combine two DESeq2 objects (dds) for analysis

Designs and contrasts

results(contrast)

  • ?results
  • Syntax: contrast=c('variable', 'numerator', 'denominator'). Example: contrast=c('Trt', 'Yes', 'No')
  • Example 1: two conditions 
    dds <- makeExampleDESeqDataSet(m=4)

dds <- DESeq(dds)
res <- results(dds, contrast=c("condition","B","A"))  # mean_B - mean_A
res
#log2 fold change (MLE): condition B vs A 
#Wald test p-value: condition B vs A 
#DataFrame with 1000 rows and 6 columns
#          baseMean log2FoldChange     lfcSE      stat    pvalue      padj
#         <numeric>      <numeric> <numeric> <numeric> <numeric> <numeric>
#gene1      2.18621      -3.029859  2.910105 -1.041151  0.297806  0.999351
#gene2     14.97050      -0.850521  1.112673 -0.764395  0.444632  0.999351
#gene3      9.31174       0.448988  1.379610  0.325446  0.744844  0.999351
#gene4     84.12136       0.333454  0.593804  0.561556  0.574419  0.999351
#gene5     21.52635      -0.538152  1.025702 -0.524667  0.599815  0.999351

counts(dds, normalized = T)[1:3, ]
#        sample1    sample2  sample3   sample4
#gene1  6.812072  0.9935038  0.00000 0.9392723
#gene2 11.677838 26.8246037 15.74392 5.6356338
#gene3  7.785225  7.9480307 18.69590 2.8178169

colData(dds)
#DataFrame with 4 rows and 2 columns
#        condition sizeFactor
#         <factor>  <numeric>
#sample1         A    1.02759
#sample2         A    1.00654
#sample3         B    1.01627
#sample4         B    1.06465

# gene 1
> mean(c(6.81, .99)) # condition A
[1] 3.9
> mean(c(0, 0.939))  # condition B
[1] 0.4695
  • Example 2: two conditions, one continuous variable (age) 
    set.seed(1234)
dds <- makeExampleDESeqDataSet(n=100,m=12)
dds$age <- rpois(12, 50)
design(dds) <- ~ condition + age
dds <- DESeq(dds) 
resultsNames(dds)
# [1] "Intercept"        "condition_B_vs_A" "age"

results(dds)  # OR  results(dds, name="age")
#log2 fold change (MLE): age 
#Wald test p-value: age 
#DataFrame with 100 rows and 6 columns
#         baseMean log2FoldChange     lfcSE       stat    pvalue      padj
#        <numeric>      <numeric> <numeric>  <numeric> <numeric> <numeric>
#gene1    2.022210    -0.04620020 0.1445006 -0.3197232  0.749178  0.920829
#gene2   24.571867     0.00360519 0.0484505  0.0744097  0.940684  0.991664
#gene3   82.491501     0.00153923 0.0329532  0.0467095  0.962745  0.991664

results(dds, contrast=c("condition","B","A")) 
# OR results(dds, name="condition_B_vs_A")

#log2 fold change (MLE): condition B vs A 
#Wald test p-value: condition B vs A 
#DataFrame with 100 rows and 6 columns
#         baseMean log2FoldChange     lfcSE      stat    pvalue      padj
#        <numeric>      <numeric> <numeric> <numeric> <numeric> <numeric>
#gene1    2.022210      3.0976656  1.854100  1.670711 0.0947788  0.910079
#gene2   24.571867     -0.1667077  0.635550 -0.262305 0.7930867  0.982211
#gene3   82.491501      0.2188290  0.431659  0.506948 0.6121910  0.982211
  • Example 3: two conditions, two genotypes, with an interaction term 
    set.seed(1234)
dds <- makeExampleDESeqDataSet(n=100,m=12)
dds$genotype <- factor(rep(rep(c("I","II"),each=3),2))

design(dds) <- ~ genotype + condition + genotype:condition
dds <- DESeq(dds) 
resultsNames(dds)
# [1] "Intercept"             "genotype_II_vs_I"     
# [3] "condition_B_vs_A"      "genotypeII.conditionB"

# the condition effect for genotype I (the main effect)
results(dds, contrast=c("condition","B","A"))

# the condition effect for genotype II
# this is, by definition, the main effect *plus* the interaction term
# (the extra condition effect in genotype II compared to genotype I).
results(dds, list( c("condition_B_vs_A","genotypeII.conditionB") ))

# the interaction term, answering: is the condition effect *different* across genotypes?
results(dds, name="genotypeII.conditionB")

results(alpha, independentFiltering)

By default alpha=.1, Set independentFiltering=FALSE if we don't like independent filtering.

Negative binomial regression and returned from results()

A matrix with columns:

  • baseMean: mean of normalized counts of all samples, normalizing for sequencing depth. It does not take into account gene length. The base mean is used in DESeq2 only for estimating the dispersion of a gene. See DESeq2 baseMean counts. 
    rowMeans(counts(dds, normalized=TRUE))
  • log2FoldChange log2FoldChange calculation in DESeq2 output, 2019 STAT115 Lect7.4 DESeq2 by Shirley Liu. Theory behind DESeq2 & Null hypothesis. Introduction to DGE from hbctraining.
    [math]\displaystyle{ \begin{align} K_{ij} & \sim NB(\mu_{ij}, \alpha_i) \\ \mu_{ij} &= s_{j} q_{ij} \\ \log_2(q_{ij}) &= x_{j} \beta_i \end{align} }[/math]
    [math]\displaystyle{ s_j }[/math] is the size factor for sample 𝑗, accounting for differences in sequencing depth.
    [math]\displaystyle{ q_{ij} }[/math] is the normalized mean count for gene 𝑖 in sample 𝑗.
    The coefficients [math]\displaystyle{ \beta_i }[/math] estimate give the log2 fold changes for gene i for each column of the model matrix [math]\displaystyle{ X }[/math].
    • In a simple linear regression [math]\displaystyle{ y = \beta_0 + \beta_1 x + \epsilon }[/math], if the covariate X takes only 0 or 1, we can show that the slope estimate [math]\displaystyle{ \frac{\sum(x_i-\bar{x})(y_i-\bar{y})}{\sum(x_i -\bar{x})^2} }[/math] can be simplified to the difference of group means [math]\displaystyle{ \bar{y}_1 - \bar{y}_0 }[/math]. This matches the idea of the fold change in gene expression.
    • You can obtain standard log fold changes (no shrinkage) by using: DESeq(dds, betaPrior=FALSE)
  • The null hypothesis being tested is: [math]\displaystyle{ H_0: \beta_i = 0 }[/math]. This tests whether the log fold change for the condition is zero, meaning there is no differential expression for gene 𝑖 between conditions.
  • lfcSE standard error value (lfcSE) returned by DeSeq2
  • Why the logFC is different between edgeR,DESeq2 results and fpkm logFC(log2(FPKM_tumor_mean/FPKM_normal_mean)? LogFCs are computed by negative binomial generalized linear models.
  • Why my log2fcs are too high?
  • stat: negative binomial GLM fitting for βi and Wald statistics by nbinomWaldTest(). log2Foldchange除以标准差所得
  • pvalue & padj (Exploring DESeq2 results: Wald test from hbctraining)

Fold-change with continuous variables

how limma work with continous variables. It's the slope/fitted coefficient of the line.

Normalization-based vs log-ratio transformation-based methods

Error with DESeq2

Error with DESeq2 “every gene contains at least one zero”

Source code

? normalizationFactors
dds <- makeExampleDESeqDataSet(n=100, m=4)

normFactors <- matrix(runif(nrow(dds)*ncol(dds),0.5,1.5),
                      ncol=ncol(dds),nrow=nrow(dds),
                      dimnames=list(1:nrow(dds),1:ncol(dds)))

normFactors <- normFactors / exp(rowMeans(log(normFactors)))
normalizationFactors(dds) <- normFactors

dds <- DESeq(dds)
res = results(dds)
res[order(res$padj), ] %>% head

class(dds)
showMethods(class="DESeqDataSet") # a long list

isS4(assays(dds))    # [1] TRUE
slotNames(assays(dds))
# [1] "listData"        "elementType"     "elementMetadata" "metadata"       
names(assays(dds))
# [1] "counts"               "normalizationFactors" "mu"  "H"    

all(counts(dds) / normFactors == counts(dds, norm=T)) # TRUE

DESeq normalization 2010 ("median ratio")

edgeR normalization

library(baySeq)
data(mobData)
head(mobData)
data(mobAnnotation)
#?mobAnnotation
head(mobAnnotation)
library(edgeR)
d <- DGEList(counts=mobData,group=factor(mobDataGroups))
d
d.full <- d # keep the old one in case we mess up
head(d.full$counts)
head(cpm(d))
apply(d$counts, 2, sum) # total gene counts per sample

d <- calcNormFactors(d)
d
  • ?calcNormFactors. If ‘object’ is a ‘matrix’, the output is a vector with length ‘ncol(object)’ giving the relative normalization factors.
y <- matrix( rpois(1000, lambda=5), nrow=200 )
calcNormFactors(y)

dge <- DGEList(M)
dge <- calcNormFactors(dge)
logCPM <- cpm(dge, log=TRUE)  # section 2.16 of vignette

DESeq2 for DE analysis

  • By default, DESeq(test = "Wald"). Another test option is LRT.
  • Vignette. Keyword: Wald test, coefficients [math]\displaystyle{ \beta }[/math], nbinomWaldTest. The coefficients βi give the log2 fold changes for gene i for each column of the model matrix X. 
    # Suppose the condition has 2 levels
dds <- DESeqDataSetFromMatrix(countData = round(counts), 
                              colData = DataFrame(condition),
                              design = ~ condition)
dds <- DESeq(dds)
res <- results(dds)
res$stat  # same as coef(dds)[, 2]/coef(dds, SE=T)[, 2]

# coef(): extracting the matrix beta_{ir} for all genes i and model coefficients r
# In the case of 2 conditions, the 1st column of coef() is intercept
#    and the 2nd column of coef() is the condition.
all(res$stat == coef(dds)[, 2]/coef(dds, SE=T)[, 2], na.rm=T )
# [1] TRUE

all(res$log2FoldChange == coef(dds)[, 2] )
all(res$lfcSE == coef(dds, SE=T)[, 2] )
  • log2FoldChange represents the log normalized counts from the mean of level 2 vs level 1. But it is not clear how to calculate that exactly. See DESEq2 log2FoldChange and lfcSE calculations.
  • According to ?results, The lfcSE gives the standard error of the log2FoldChange. For the Wald test, stat is the Wald statistic: the log2FoldChange divided by lfcSE, which is compared to a standard Normal distribution to generate a two-tailed pvalue.
  • Differential expression analysis with DESeq2
  • Videos from Chipster Tutorials https://www.youtube.com/watch?v=5tGCBW3_0IA 
    > samplesDESeq = with(samples, data.frame(
         shortname = I(shortname),
         countf = I(countf),
         condition = condition,
         LibraryLayout = LibraryLayout))

> samplesDESeq

 shortname              countf condition LibraryLayout
1   CT.PA.1   Untreated-3.count       CTL        PAIRED
2   CT.PA.2   Untreated-4.count       CTL        PAIRED
3   KD.PA.3 CG8144_RNAi-3.count        KD        PAIRED
4   KD.PA.4 CG8144_RNAi-4.count        KD        PAIRED
5   CT.SI.5   Untreated-1.count       CTL        SINGLE
6   KD.SI.6 CG8144_RNAi-1.count        KD        SINGLE
7   CT.SI.7   Untreated-6.count       CTL        SINGLE

> library("DESeq")
cds <- newCountDataSetFromHTSeqCount(samplesDESeq) # quick, 15682 features, 7 samples
cds <- estimateSizeFactors(cds) # quick
sizeFactors(cds)

#   CT.PA.1   CT.PA.2   KD.PA.3   KD.PA.4   CT.SI.5   KD.SI.6   CT.SI.7
# 0.6991612 0.8104921 0.8217403 0.8941097 1.6431467 1.3720872 1.1041186

cds <- estimateDispersions(cds) # quick
res <- nbinomTest(cds,"CTL","KD") # 44 seconds
sum(res$pval <= .05, na.rm=T) # [1] 1574
sum(res$padj <= .05, na.rm=T) # [1] 730

options(width=100)
res[1:3,]
#            id  baseMean baseMeanA baseMeanB foldChange log2FoldChange      pval padj
# 1 FBgn0000003  0.000000  0.000000  0.000000        NaN            NaN        NA   NA
# 2 FBgn0000008 88.526695 88.486694 88.580029   1.001055    0.001520941 0.9298627    1
# 3 FBgn0000014  3.054418  2.327533  4.023599   1.728697    0.789684778 0.6839858    1

DESeqDataSet methods

  • counts(): This method returns the raw count data for the genes in the DESeqDataSet object.
  • colData(): This method returns the metadata (e.g., sample information) for the samples in the DESeqDataSet object.
  • rowRanges()
  • design()
  • sizeFactors(): This method returns the size factors for the DESeqDataSet object, which are used to adjust for library size differences between samples.
  • rld(): This method returns the regularized-logarithm (rlog) transformed data for the DESeqDataSet object, which is a variance-stabilizing transformation that can be used for visualization and clustering.
  • plotDispEsts(): This method generates a plot of the dispersion estimates for the DESeqDataSet object, which can be used to diagnose the fit of the model.
  • plotCounts(): This method generates a plot of the raw count data for the DESeqDataSet object, which can be used to visualize the distribution of the data.
  • results(): This method returns the results of the differential expression analysis for the DESeqDataSet object, including the log2FoldChange values and the p-values.
  • resultsNames()
  • coef()
  • plotMA(): MA plot
  • Volcano plot:
    • EnhancedVolcano::EnhancedVolcano(). Note:
      1. There are no default values for x and y parameters. All examples in the vignette use y = ‘pvalue’ (not ‘padj’).
      2. pCutoff means a p-value, not fdr adjustment
      EnhancedVolcano(res,
    lab = rownames(res),
    pCutoff = 1e-05,
    FCcutoff = 1.0,
    x = 'log2FoldChange',
    y = 'pvalue')

      To draw adjusted p-values on the y-axis and use adjusted p-values as a cutoff (however, the label for some sig genes are missing. IOW, ggplot2 solution is better) 

      EnhancedVolcano(res,
    lab = rownames(res),
    pCutoff = 0.05,
    pCutoffCol = "padj", 
    FCcutoff = 1.0,
    ylim = c(0, max(-log10(res$padj), na.rm = TRUE)),
    labSize = 2.5,
    x = 'log2FoldChange',
    y = 'padj',
    title = 'Volcano plot',
    ylab = bquote(~-Log[10]~'Adjusted p-value'),
    legendLabels = c('NS', 'Log2 FC', 'Adjusted p-value', 'Adjusted p-value & Log2 FC'))
    • ggplot2 
      ggplot(res, aes(x = log2FoldChange, y = -log10(pvalue))) + 
  geom_point() +
  xlab("log2 Fold Change") + ylab("-log10(p-value)") +
ggtitle("Volcano Plot")

      If we like to add vertical and horizontal cutoff lines and change to use adjusted p-values (padj column), we can follow Making Volcano Plots With ggplot2: 

      library(ggplot2)
library(ggrepel)

data <- res
data$gene <- rownames(res)
ggplot(data, aes(x = log2FoldChange, y = -log10(padj))) +
    geom_point(aes(color = padj < 0.05 & abs(log2FoldChange) > 1), alpha = 0.5) +
    scale_color_manual(values = c("black", "red"), na.translate = F) +
    geom_vline(
      xintercept = c(-1, 1),
      linetype = 'dashed'
    ) +
    geom_hline(
      yintercept = -log10(.05),
      linetype = 'dashed'
    ) +
    theme_bw() +
    labs(title = "Volcano Plot", x = "Log2 Fold Change", y = "-Log10 Adjusted P-Value") +
    geom_label_repel(
        data = significant_genes,
        aes(label = gene),
        size=3,
        box.padding = 0.25,     # default
        point.padding = 1e-06,  # default
        max.overlaps = 10       # default
    )

Pre-filter

betaPrior is FALSE by default

Different results in DESeq2 when using betaPrior=TRUE and betaPrior=FALSE

lfcShrink

Getting NAs in p-values

Getting NAs in both p-values (normal and adjusted) in DEseq2

Comparison

A comparison study on modeling of clustered and overdispersed count data for multiple comparisons Kruppa 2020

Wald vs Likelihood ratio test

  • HELP - DESeq2 difference between LRT and Wald Test in RNA-seq/ATAC-seq.
    • The Wald tests are specific to a given contrast. You are specifically asking if there is evidence that the coefficient comparing two groups is larger than zero... I would imagine that the vast majority of people use Wald tests exclusively, as they are mostly wanting to make specific comparisons.
    • If you fit a full and reduced model and then compute a likelihood ratio test, you are testing if the full model fits the data better than the reduced model. It's an omnibus test that tells you that at least one coefficient is significant, much like an F-test in a conventional linear regression.

DESeq2, edgeR, limma

DESeq2 paired multifactor test

DESeq2 paired multifactor test

t-test vs DESeq2

FPKM t-test vs DEseq2. t-tests are parametric, based on the normal distribution. t-tests are expected to yield far fewer DEGs simply because at low sample size they're massively underpowered, please read papers to learn why. That is the basis why methods such as DESeq2 even exist. RNA-seq is not normally distributed, that is well-known.

Time series

Interaction

Complex DESeq2 Experimental Design

File Format

fastq

The wikipedia website provides information to convert FASTQ to FASTA format (when do we want to do that?).

fastq format

A FASTQ file normally uses four lines per sequence. 

@SEQ_ID
GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT
+
!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65
  • Line 1 begins with a '@' character and is followed by a sequence identifier and an optional description (like a FASTA title line).
  • Line 2 is the raw sequence letters.
  • Line 3 begins with a '+' character and is optionally followed by the same sequence identifier (and any description) again.
  • Line 4 encodes the quality values for the sequence in Line 2, and must contain the same number of symbols as letters in the sequence.

It seems line 3 & 4 are not required. See the simulated fastq files on http://genomespot.blogspot.com/2014/11/dna-aligner-accuracy-bwa-bowtie-soap.html. I can use BWA to run alignment without a problem:)

Paired end

Forward and reverse reads in paired-end sequencing

The paired files can be two formats. See Bioinformatics at COMAV

Format1: 

Fastq file 1
@molecule_1 1st_read_from_pair
@molecule_2 1st_read_from_pair
@molecule_3 1st_read_from_pair

Fastq file 2
@molecule_1 2nd_read_from_pair
@molecule_2 2nd_read_from_pair
@molecule_3 2nd_read_from_pair

Format 2: 

Interleaved Fastq file
@molecule_1 1st_read_from_pair
@molecule_1 2nd_read_from_pair
@molecule_2 1st_read_from_pair
@molecule_2 2nd_read_from_pair
@molecule_3 1st_read_from_pair
@molecule_3 2nd_read_from_pair

Note from my experiment, if we flip the two fastq files order in the BWA command, the resulting bam file will diff at FLAG (2nd) column. The final vcf file is not changed.

FastQValidator

http://genome.sph.umich.edu/wiki/FastQValidator

fasta

FASTA format is a text-based format for representing nucleotide sequences . cf. FASTQ files provide more information than FASTA since FASTQ format stores both a biological sequence (usually nucleotide sequence) and its corresponding quality scores.

The reference genome is saved in FASTA format.

Normally the first line in a FASTA file starts with a ">" (greater-than) symbol. Subsequent lines starting with a semicolon would be ignored by software. Following the initial line (used for a unique description of the sequence) is the actual sequence itself in standard one-letter code. A multiple sequence FASTA format would be obtained by concatenating several single sequence FASTA files.

For the fruit fly genome file Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa. The grep -n command can be used to get the row numbers of some keyword (chromosome names in this case). 

$ head Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa
>2L dna:chromosome chromosome:BDGP5:2L:1:23011544:1 REF
CGACAATGCACGACAGAGGAAGCAGAACAGATATTTAGATTGCCTCTCATTTTCTCTCCC
ATATTATAGGGAGAAATATGATCGCGTATGCGAGAGTAGTGCCAACATATTGTGCTCTTT
GATTTTTTGGCAACCCAAAATGGTGGCGGATGAACGAGATGATAATATATTCAAGTTGCC
GCTAATCAGAAATAAATTCATTGCAACGTTAAATACAGCACAATATATGATCGCGTATGC
GAGAGTAGTGCCAACATATTGTGCTAATGAGTGCCTCTCGTTCTCTGTCTTATATTACCG
CAAACCCAAAAAGACAATACACGACAGAGAGAGAGAGCAGCGGAGATATTTAGATTGCCT
ATTAAATATGATCGCGTATGCGAGAGTAGTGCCAACATATTGTGCTCTCTATATAATGAC
TGCCTCTCATTCTGTCTTATTTTACCGCAAACCCAAATCGACAATGCACGACAGAGGAAG
CAGAACAGATATTTAGATTGCCTCTCATTTTCTCTCCCATATTATAGGGAGAAATATGAT

$ grep -n ">" Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa
1:>2L dna:chromosome chromosome:BDGP5:2L:1:23011544:1 REF
383528:>2LHet dna:chromosome chromosome:BDGP5:2LHet:1:368872:1 REF
389677:>2R dna:chromosome chromosome:BDGP5:2R:1:21146708:1 REF
742124:>2RHet dna:chromosome chromosome:BDGP5:2RHet:1:3288761:1 REF
796938:>3L dna:chromosome chromosome:BDGP5:3L:1:24543557:1 REF
1205999:>3LHet dna:chromosome chromosome:BDGP5:3LHet:1:2555491:1 REF
1248592:>3R dna:chromosome chromosome:BDGP5:3R:1:27905053:1 REF
1713678:>3RHet dna:chromosome chromosome:BDGP5:3RHet:1:2517507:1 REF
1755638:>4 dna:chromosome chromosome:BDGP5:4:1:1351857:1 REF
1778170:>U dna:chromosome chromosome:BDGP5:U:1:10049037:1 REF
1945655:>Uextra dna:chromosome chromosome:BDGP5:Uextra:1:29004656:1 REF
2429067:>X dna:chromosome chromosome:BDGP5:X:1:22422827:1 REF
2802782:>XHet dna:chromosome chromosome:BDGP5:XHet:1:204112:1 REF
2806185:>YHet dna:chromosome chromosome:BDGP5:YHet:1:347038:1 REF
2811970:>dmel_mitochondrion_genome dna:chromosome chromosome:BDGP5:dmel_mitochondrion_genome:1:19517:1 REF

$ wc -l *.fa
2812296 Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa

For human, 

$ grep -n ">" ~/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa
1:>chrM
334:>chr1
4985348:>chr2
9849337:>chr3
13809787:>chr4
17632874:>chr5
21251181:>chr6
24673484:>chr7
27856259:>chr8
30783541:>chr9
33607811:>chr10
36318507:>chr11
39018639:>chr12
41695678:>chr13
43999077:>chr14
46146069:>chr15
48196698:>chr16
50003795:>chr17
51627701:>chr18
53189247:>chr19
54371828:>chr20
55632340:>chr21
56594939:>chr22
57621032:>chrX
60726445:>chrY

chromosome names

When I apply the above method to examine the genome.fa files from Ensembl, NCBI and UCSC using the command 

grep -n ">" genome.fa | cut -d' ' -f1 | cut -d '>' -f2
# for example
grep -n ">" ~/Downloads/Homo_sapiens.GRCh38.dna.chromosome.22.fa | cut -d' ' -f1 | cut -d '>' -f2  
# 22

I get the following result

  1. Ensembl:
    1. GRCh37: 1, 2, 3, ...., X, Y, MT
    2. GRCh38: 1, 2, 3, ... (see note below)
  2. NCBI:
    1. build37.1, build37.2: 1, 2, 3, ....,X, Y, MT
    2. GRCh38: chr1, chr2, ...., chr22, chrX, chrY, chrM, chr1_KI270706v1_random, ....., chrEBV
  3. UCSC:
    1. hg18, hg19: chr1, chr2, ...., chr22, chrX, chrY, chrM
    2. hg38: chr1, chr2, ..., chr22, chrX, chrY, chrM, ..., chrUn_GL000218v1, chrEBV

Note that

  • Neither Illumina or Biowulf provide GRCh38 from Ensembl (maybe for a good reason). Ensembl (http://ftp.ensembl.org/pub/release-76/fasta/homo_sapiens/dna) does provide GRCh38.
  • GRCh38 from Ensembl is still following its GRCh37 convention; it won't include 'chr' on their chromosome names. That is, even the same name GRCh38 was used in both Ensembl and NCBI, they have different conventions.

chromosome sizes

https://www.biostars.org/p/134807/ 

GENOMEFA=Arabidopsis_thaliana.TAIR10.23.dna.genome.fa

# Method 1: use pyfaidx
# upgrade pip from 8.1.2 to 9.0.1
sudo pip install --upgrade pip
sudo pip install pyfaidx
$ cd /tmp
$ faidx $GENOMEFA -i chromsizes > sizes.genome
$ cat sizes.genome
Pt	154478
Mt	366924
4	18585056
2	19698289
3	23459830
5	26975502
1	30427671
# Method 2: use samtools
$ /opt/SeqTools/bin/samtools-1.3/samtools faidx $GENOMEFA
$ cat $GENOMEFA.fai
Pt	154478	51	60	61
Mt	366924	157155	60	61
4	18585056	530246	60	61
2	19698289	19425104	60	61
3	23459830	39451749	60	61
5	26975502	63302628	60	61
1	30427671	90727773	60	61
$ cut -f1,2 $GENOMEFA.fai > sizes.genome
$ cat sizes.genome2
Pt	154478
Mt	366924
4	18585056
2	19698289
3	23459830
5	26975502
1	30427671

R approach 

library(Biostrings)
x <- readDNAStringSet("~/igenomes/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa")
y <- width(x[1:24])
barplot(y, las = 2)
y
#     chr1      chr2      chr3      chr4      chr5      chr6      chr7      chr8 
# 248956422 242193529 198295559 190214555 181538259 170805979 159345973 145138636 
#      chr9     chr10     chr11     chr12     chr13     chr14     chr15     chr16 
# 138394717 133797422 135086622 133275309 114364328 107043718 101991189  90338345 
#     chr17     chr18     chr19     chr20     chr21     chr22      chrX      chrY 
#  83257441  80373285  58617616  64444167  46709983  50818468 156040895  57227415

Chromosome len.png

Remove/Add 'chr' from fasta

sed 's/chr//g' ucsc.hg19.fa >ucsc.hg19.nochr.fa
# or
cat hg19.fa | sed 's/>chr/>/g' > hg19_new.fa

Similarly to add 'chr' to fasta: 

cat hg19.fa | sed 's/>/>chr/g' > hg19_new.fa

Convert Fasta to Fastq

It is OK to use Fasta file as the input sequencing files in alignment programs.

If we still want to convert FASTA to FASTQ, two methods are given with an example here. Note quality scores are padded.

Convert Fastq to Fasta

Note the quality scores are discarded. 

$ cat test.fastq
@SRR002051.3554692 :8:290:671:993 length=33
CCATTCCCCAAAACAGTAAAACCACACACCGAG
+SRR002051.3554692 :8:290:671:993 length=33
40&&+(&#$&3""%$#"#%$)%"%"%$/%%""#
@SRR002051.3554693 :8:290:941:550 length=33
ACTAACATCCCATAATCGTACCTCCTACCATCA
+SRR002051.3554693 :8:290:941:550 length=33
IIII*I$+(/II'$&:I"%""""&"&$%"'""I
$ cat test.fastq | paste - - - - | sed 's/^@/>/g'| cut -f1-2 | tr '\t' '\n'
>SRR002051.3554692 :8:290:671:993 length=33
CCATTCCCCAAAACAGTAAAACCACACACCGAG
>SRR002051.3554693 :8:290:941:550 length=33
ACTAACATCCCATAATCGTACCTCCTACCATCA
$ head simulated_reads_testbeers.fa
>seq.1a
CGAAGAAGGACCCAAAGATGACAAGGCTCACAAAGTACACCCAGGGCAGTTCATACCCCATGGCATCTTGCATCCAGTAGAGCACATCGGTCCAGCCTTC
>seq.1b
GCTCGAGCTGTTCCTTGGACGAATGCACAAGACGTGCTACTTCCTGGGATCCGACATGGAAGCGGAGGAGGACCCATCGCCCTGTGCATCTTCGGGATCA
>seq.2a
GCCCCAGCAGAGCCGGGTAAAGATCAGGAGGGTTAGAAAAAATCAGCGCTTCCTCTTCCTCCAAGGCAGCCAGACTCTTTAACAGGTCCGGAGGAAGCAG
>seq.2b
ATGAAGCCTTTTCCCATGGAGCCATATAACCATAATCCCTCAGAAGTCAAGGTCCCAGAATTCTACTGGGATTCTTCCTACAGCATGGCTGATAACAGAT
>seq.3a
CCCCAGAGGAGCGCCACCTGTCCAAGATGCAGCAGAACGGCTACGAAAATCCAACCTACAAGTTCTTTGAGCAGATGCAGAACTAGACCCCCGCCACAGC
$ awk 'NR % 4 == 1 {print ">" $0 } NR % 4 == 2 {print $0}' simulated_reads_testbeers.fa > my.fasta
$ head my.fasta
>>seq.1a
CGAAGAAGGACCCAAAGATGACAAGGCTCACAAAGTACACCCAGGGCAGTTCATACCCCATGGCATCTTGCATCCAGTAGAGCACATCGGTCCAGCCTTC
>>seq.2a
GCCCCAGCAGAGCCGGGTAAAGATCAGGAGGGTTAGAAAAAATCAGCGCTTCCTCTTCCTCCAAGGCAGCCAGACTCTTTAACAGGTCCGGAGGAAGCAG
>>seq.3a
CCCCAGAGGAGCGCCACCTGTCCAAGATGCAGCAGAACGGCTACGAAAATCCAACCTACAAGTTCTTTGAGCAGATGCAGAACTAGACCCCCGCCACAGC
>>seq.4a
CGGGTGGAGGCTGGCCAAGCAACGAAGGAGATAAAATTGCAGCTGGCATTGTGTGTGCGCGCGCCCGCGGCGGCGGCCAGCAGGGGTGCGCGCGGAGCGG
>>seq.5a
CGCTCACCTTCAATGTCACTGGCAAACATACTGCCTACCAGCTACTCCTCAGCCTTACACCTTAAGCATACGTCCTGCTCTTACTCTTCGAGTTGAGGGT
$ awk 'NR % 4 == 0 {print ">" $0 } NR % 4 == 3 {print $0}' simulated_reads_testbeers.fa > my.fasta
$ head my.fasta
>seq.1b
>GCTCGAGCTGTTCCTTGGACGAATGCACAAGACGTGCTACTTCCTGGGATCCGACATGGAAGCGGAGGAGGACCCATCGCCCTGTGCATCTTCGGGATCA
>seq.2b
>ATGAAGCCTTTTCCCATGGAGCCATATAACCATAATCCCTCAGAAGTCAAGGTCCCAGAATTCTACTGGGATTCTTCCTACAGCATGGCTGATAACAGAT
>seq.3b
>GTTTGATACAGCTAAATTCTTTACAGTACACAAAACCCATTAATAATGTAGTATAGAGACCAAAATGTAAAGAGAGATAGAATACATTACTGATGTGTGG
>seq.4b
>CGGGGCCCCGCCCGCGGCTAGGGAGGTGGCCGCCCTGGCCCGAGCCTGCTGCCCACGGCCGCGGCACTCAGCTCGCACTCCTTCAGCCCGGGAGGCCGGG
>seq.5b
>ACTCCCTGATAGTCTATGGAAGGAAAATGACAACTATTTTAGAATATTTCTAGTTTGTTTTTTCAGTGATCTTTTCATCCAGGCCTTGTTACTGTTACAG

GATK

When I use Ensembl/GRCh37 as the reference to run GATK, I got an error 

##### ERROR MESSAGE: Input files /home/brb/SeqTestdata/usefulvcf/hg19/gatk/common_all_20160601.vcf and reference have incompatible contigs. Please see http://gatkforums.broadinstitute.org/discussion/63/input-files-have-incompatible-contigsfor more information. Error details: No overlapping contigs found.
##### ERROR   /home/brb/SeqTestdata/usefulvcf/hg19/gatk/common_all_20160601.vcf contigs = [chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22, chrX, chrY]
##### ERROR   reference contigs = [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, Y, MT]

The conclusion is I need to check if the chromosome name in dbsnp vcf file (ftp://ftp.ncbi.nih.gov/snp/organisms/) and reference fa file has the same naming convention.

For example, for Ensemble/GRCh37, I should NOT use the GATK version (i.e. no 'chr' was added) from ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh37p13/VCF/

(P.S. For UCSC hg19, we shall use the GATK version ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh37p13/VCF/GATK/)

But for UCSC/hg38, I should use the GATK version ('chr' was added) from ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606_b147_GRCh38p2/VCF/GATK/. Read the readme

So I end up with the file structure in my PC

  • usefulvcf/hg19/common_all*.vcf (no 'chr' was added)
  • usefulvcf/hg38/gatk/common_all*.vcf usefulvcf/hg38/chr/common_all*.vcf ('chr' was added)

gff/gtf

brb@T3600 ~ $ ls -l ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/
total 4
drwxrwxr-x 9 brb brb 4096 Jul 24  2015 Archives
lrwxrwxrwx 1 brb brb   30 Mar 10 03:03 Genes -> Archives/archive-current/Genes
lrwxrwxrwx 1 brb brb   35 Mar 10 03:03 README.txt -> Archives/archive-current/README.txt
lrwxrwxrwx 1 brb brb   33 Mar 10 03:03 SmallRNA -> Archives/archive-current/SmallRNA
lrwxrwxrwx 1 brb brb   34 Mar 10 03:03 Variation -> Archives/archive-current/Variation
brb@T3600 ~ $ ls -lH ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Archives
total 28
drwxrwxr-x 4 brb brb 4096 Mar 15  2012 archive-2010-09-27-22-25-17
drwxrwxr-x 5 brb brb 4096 Mar 15  2012 archive-2011-01-27-18-25-49
drwxrwxr-x 5 brb brb 4096 Mar 15  2012 archive-2011-08-30-21-45-18
drwxrwxr-x 5 brb brb 4096 Mar 15  2012 archive-2012-03-09-03-24-41
drwxrwxr-x 5 brb brb 4096 Oct  1  2013 archive-2013-03-06-11-23-03
drwxrwxr-x 5 brb brb 4096 Jun  2  2014 archive-2014-06-02-13-47-56
drwxrwxr-x 5 brb brb 4096 Jul 24  2015 archive-2015-07-17-14-32-32
lrwxrwxrwx 1 brb brb   27 Mar 10 03:35 archive-current -> archive-2015-07-17-14-32-32
  • GTF file is used in RNA-Seq reads alignment and count reads.
    • Tophat - If this option is provided, TopHat will first extract the transcript sequences and use Bowtie to align reads to this virtual transcriptome first. Only the reads that do not fully map to the transcriptome will then be mapped on the genome. The reads that did map on the transcriptome will be converted to genomic mappings (spliced as needed) and merged with the novel mappings and junctions in the final tophat output.
    • STAR - GTF file (containing annotated transcripts) can be used in creating STAR index files (--sjdbGTFfile option in STAR --runMode genomeGenerate). STAR will extract splice junctions from this file and use them to greatly improve accuracy of the mapping. Note that in the mapping step there is no need to supply the GTF file again.
    • htseq-count: one of two required inputs (the other one is a sorted bam file).
  • The meaning of Gene isoform in wikipedia. It contains links to TSS, CDS, UTR.
  • The meaning of transcriptome in wikipedia. Each row in a GTF file contains one feature and the the feature's transcript id and gene name. See gist.
  • Gene name in GTF file
$ grep KRAS ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf | wc -l
23

The following output shows how many features from different sources. 

brb@brb-T3500:/tmp$ wc -l ~/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa
61913917 /home/brb/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa
brb@brb-T3500:/tmp$ wc -l ~/igenomes/Homo_sapiens/NCBI/build37.2/Sequence/WholeGenomeFasta/genome.fa
44224234 /home/brb/igenomes/Homo_sapiens/NCBI/build37.2/Sequence/WholeGenomeFasta/genome.fa
brb@brb-T3500:/tmp$ wc -l ~/igenomes/Homo_sapiens/Ensembl/GRCh37/Sequence/WholeGenomeFasta/genome.fa
51594937 /home/brb/igenomes/Homo_sapiens/Ensembl/GRCh37/Sequence/WholeGenomeFasta/genome.fa

brb@brb-T3500:~/igenomes/Homo_sapiens$ wc -l UCSC/hg19/Annotation/Genes/genes.gtf
869204 UCSC/hg19/Annotation/Genes/genes.gtf
brb@brb-T3500:~/igenomes/Homo_sapiens$ wc -l NCBI/build37.2/Annotation/Genes/genes.gtf
819119 NCBI/build37.2/Annotation/Genes/genes.gtf
brb@brb-T3500:~/igenomes/Homo_sapiens$ wc -l Ensembl/GRCh37/Annotation/Genes/genes.gtf
2280612 Ensembl/GRCh37/Annotation/Genes/genes.gtf

An example from UCSC/hg19. As you can see from IGV and the output of the first few rows,

  • the locations of each rows are not exclusive. For example, rows #3 and #4 are overlapped that matches with what we saw on IGV (fly example)
  • one row represents one feature; one transcript consists several features
  • one gene (e.g. WASH7P) can be split over multiple regions (UCSC/hg19 example).
$ more ~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf
chr1    unknown exon    11874   12227   .       +       .       gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS16932";
chr1    unknown exon    12613   12721   .       +       .       gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS16932";
chr1    unknown exon    13221   14409   .       +       .       gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS16932";
chr1    unknown exon    14362   14829   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    14970   15038   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    15796   15947   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    16607   16765   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    16858   17055   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    17233   17368   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    17369   17436   .       -       .       gene_id "MIR6859-3"; gene_name "MIR6859-3"; transcript_id "NR_107063_2"; tss_id "TSS25677";
chr1    unknown exon    17369   17436   .       -       .       gene_id "MIR6859-2"; gene_name "MIR6859-2"; transcript_id "NR_107062_1"; tss_id "TSS25677";
chr1    unknown exon    17369   17436   .       -       .       gene_id "MIR6859-4"; gene_name "MIR6859-4"; transcript_id "NR_128720_2"; tss_id "TSS25677";
chr1    unknown exon    17369   17436   .       -       .       gene_id "MIR6859-1"; gene_name "MIR6859-1"; transcript_id "NR_106918_2"; tss_id "TSS25677";
chr1    unknown exon    17606   17742   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    17915   18061   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    18268   18366   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    24738   24891   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    29321   29370   .       -       .       gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS8568";
chr1    unknown exon    30366   30503   .       +       .       gene_id "MIR1302-11"; gene_name "MIR1302-11"; transcript_id "NR_036268"; tss_id "TSS10004";

Igv hg19.png

The following table gives an example (UCSC/hg19) from one feature. Notice that the last column contains transcript ID, tss ID and gene ID. If the GTF is obtained from Ensembl, this column also has exon ID.

1 seqname chr1 (called 1 from NCBI & Ensembl)
2 source unknown
3 feature exon/transcript
4 start 11874
5 end 12227
6 score .
7 strand +
8 frame 0
9 attribute gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS14844";

The gene attributes (gene_id, transcript_id, exon_number, gene_name, gene_biotype, transcript_name, protein_id) appears on the mouse-over pop-up in IGV. See the 1st screenshot below from fly genome (imported manually by myself to IGV). From the 1st screenshot it is clear that one position may contain more than one transcripts.

IGV fly Anders2013.png IGV fly Anders2013 2.png

This post on seqanswers.com has a discussion about the GTF file, exon, intron, CDS, exon, etc. It says

"Exon" refers to transcription (DNA->RNA) and "CDS" to translation (RNA->Proteins). These are two different biological mechanisms.

rtracklayer package

See also the vignette of SingscoreAMLMutations 

brb@brb-T3500:~/igenomes/Homo_sapiens$ R
> x <- read.delim("~/igenomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf", header=FALSE, as.is=TRUE)
> dim(x)
[1] 2280612       9
> x[1:2, 1:8]
  V1                   V2         V3    V4    V5 V6 V7 V8
1  1 processed_transcript       exon 11869 12227  .  +  .
2  1 processed_transcript transcript 11869 14409  .  +  .
> strsplit(x[1:2, 9], ";") # not the same number of elements so we cannot apply as.data.frame()
[[1]]
 [1] "exon_id ENSE00002234944"        " exon_number 1"
 [3] " gene_biotype pseudogene"       " gene_id ENSG00000223972"
 [5] " gene_name DDX11L1"             " gene_source ensembl_havana"
 [7] " transcript_id ENST00000456328" " transcript_name DDX11L1-002"
 [9] " transcript_source havana"      " tss_id TSS15145"

[[2]]
[1] "gene_biotype pseudogene"        " gene_id ENSG00000223972"
[3] " gene_name DDX11L1"             " gene_source ensembl_havana"
[5] " transcript_id ENST00000456328" " transcript_name DDX11L1-002"
[7] " transcript_source havana"      " tss_id TSS15145"

> table(x[, 3])

        CDS        exon start_codon  stop_codon
     794920     1309155       92839       83698
> y <- read.delim("~/igenomes/Homo_sapiens/NCBI/build37.2/Annotation/Genes/genes.gtf", header=FALSE, as.is=TRUE)
> table(y[, 3])

        CDS        exon start_codon  stop_codon
     345395      405671       34041       34012
> z <- read.delim("~/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf", header=FALSE, as.is=TRUE)
> table(z[, 3])

        CDS        exon start_codon  stop_codon
     365947      430178       36547       36532

> options(width=120)
> table(x[, 2])

          3prime_overlapping_ncrna                          antisense                          IG_C_gene
                                92                              40326                                290
                   IG_C_pseudogene                          IG_D_gene                          IG_J_gene
                                40                                196                                 78
                   IG_J_pseudogene                          IG_V_gene                    IG_V_pseudogene
                                12                               1099                                657
                           lincRNA                              miRNA                           misc_RNA
                             48588                               6848                               4380
                           Mt_rRNA                            Mt_tRNA            nonsense_mediated_decay
                                 4                                 44                             293471
                    non_stop_decay             polymorphic_pseudogene               processed_pseudogene
                              1132                               1853                              24516
              processed_transcript                     protein_coding                         pseudogene
                            172582                            1978244                               2247
                   retained_intron                               rRNA                     sense_intronic
                            150034                               1140                               2474
                 sense_overlapping                             snoRNA                              snRNA
                              1255                               3242                               4148
  transcribed_processed_pseudogene transcribed_unprocessed_pseudogene    translated_processed_pseudogene
                              1182                               7931                                  3
                         TR_C_gene                          TR_D_gene                          TR_J_gene
                                58                                  9                                246
                   TR_J_pseudogene                          TR_V_gene                    TR_V_pseudogene
                                 8                                869                                107
                unitary_pseudogene             unprocessed_pseudogene
                              1467                              13763
> table(y[, 2])

unknown
 819119
> table(z[, 2])

unknown
 869204

Another example. Read GTF file into R. 

library(rtracklayer)
x <- import("/fdb/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf")
x
# GRanges object with 1006819 ranges and 9 metadata columns:
length(unique(x$gene_name))
# [1] 26364
length(unique(x$transcript_id))
# [1] 54070

y <- read.delim("/fdb/igenomes/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf")
dim(y)
# [1] 1006818       9
colnames(y)
# [1] "chr1"
# [2] "unknown"
# [3] "exon"
# [4] "X11874"
# [5] "X12227"
# [6] "."
# [7] "X."
# [8] "..1"
# [9] "gene_id.DDX11L1..gene_name.DDX11L1..transcript_id.NR_046018..tss_id.TSS16932."

Convert transcript to symbol

  • Check out XXX.genes.results or XXX.isoforms.results file from RSEM output 
    $ head Sample1.genes.results
gene_id	transcript_id(s)	length	effective_length	expected_count	TPM	FPKM
A1BG	NM_130786	1766.00	1589.99	0.00	0.00	0.00
A1BG-AS1	NR_015380	2130.00	1953.99	0.00	0.00	0.00

$ head Sample1.isoforms.results
transcript_id	gene_id	length	effective_length	expected_count	TPM	FPKM	IsoPct
NM_130786	A1BG	1766	1589.99	0.00	0.00	0.00	0.00
NR_015380	A1BG-AS1	2130	1953.99	0.00	0.00	0.00	0.00
  • biotools (online)
  • Converting between UCSC id and gene symbol with bioconductor annotation resources. You need to use the Homo.sapiens package. 
    library(Homo.sapiens)
select(Homo.sapiens, "uc001qvk.1", "SYMBOL","TXNAME")
# 'select()' returned 1:1 mapping between keys and columns
#       TXNAME SYMBOL
# 1 uc001qvk.1    A2M
  • How to map UCSC transcripts to gene symbol? org.Hs.eg.db package.

Splice junction

GTF file contains splice junction information. See Genome > Splice junction on how to extract this information.

bed

A BED file (.bed) is a tab-delimited text file that defines a feature track. Bed files can be like GTF/GFF to provide gene annotations. See ensembl.org. Tracks in the UCSC Genome Browser (http://genome.ucsc.edu/) can be downloaded to BED files and loaded into IGV.

A simple bed file looks like 

chr7    127471196  127472363

The format is explained in UCSC.

We can also take a look some bed files generated after running the Tophat program. 

brb@brb-T3500:~/Anders2013/Untreated-6$ head -n 3 insertions.bed
track name=insertions description="TopHat insertions"
2L      10457   10457   T       1
2L      10524   10524   T       1
brb@brb-T3500:~/Anders2013/Untreated-6$ head -n 3 deletions.bed
track name=deletions description="TopHat deletions"
2L      10832   10833   -       1
2L      12432   12434   -       1
brb@brb-T3500:~/Anders2013/Untreated-6$ head -n 3 junctions.bed
track name=junctions description="TopHat junctions"
2L      11095   11479   JUNC00000001    4       -       11095   11479   255,0,0 2       74                                           ,70     0,314
2L      11271   11479   JUNC00000002    24      -       11271   11479   255,0,0 2       73                                           ,70     0,138

From my observations, the IGV can create the junctions track (it is called 'Untreated-6_s.bam junctions' when I load the <Untreated-6_s.bam> file) automatically once we load the bam file. If we manually load the <junctions.bed> file saved in the 'Untreated-6' directory, we can see both junction tracks are the same.

sam/bam, "samtools view" and Rsamtools

There are 11 required fields in the sam file and the header is optional. See also http://bio-bwa.sourceforge.net/bwa.shtml.

column field example (/ExomeLungCancer/H1975cellLine)
1 QNAME SRR2923335.1
2 FLAG 99
3 RNAME (chromosome name) 6
4 POS 36938157
5 MAPQ 60
6 CIGAR 100M
7 RNEXT =
8 PNEXT 36938259
9 TLEN 202
10 SEQ NGCAAGTAGAGGGGCGCACACCTACCCGCAGTTGTTCACAGCCATG…
11 QUAL #1=DDFDFHHHHHJIJJJJIJJJJJJJJIJJJJJIJIHHHHHFFF…
12 Tags (optional) NM:i:1 MD:Z:0G99 AS:i:99 XS:i:61 MQ:i:60

We can use samtools view to view the bam file (samtools view will not include the header, samtools view -H will include only the header, samtools view -h will include the header) 

$ samtools view -h accepted_hits.bam | more
@HD	VN:1.4
@SQ	SN:1	LN:249250621
@SQ	SN:2	LN:243199373
@SQ	SN:3	LN:198022430
@SQ	SN:4	LN:191154276
@SQ	SN:5	LN:180915260
@SQ	SN:6	LN:171115067
@SQ	SN:7	LN:159138663
@SQ	SN:8	LN:146364022
@SQ	SN:9	LN:141213431
@SQ	SN:10	LN:135534747
@SQ	SN:11	LN:135006516
@SQ	SN:12	LN:133851895
@SQ	SN:13	LN:115169878
@SQ	SN:14	LN:107349540
@SQ	SN:15	LN:102531392
@SQ	SN:16	LN:90354753
@SQ	SN:17	LN:81195210
@SQ	SN:18	LN:78077248
@SQ	SN:19	LN:59128983
@SQ	SN:20	LN:63025520
@SQ	SN:21	LN:48129895
@SQ	SN:22	LN:51304566
@SQ	SN:X	LN:155270560
@SQ	SN:Y	LN:59373566
@SQ	SN:MT	LN:16569
@PG	ID:STAR	PN:STAR	VN:STAR_2.5.0c	CL:STAR   --runThreadN 11   --genomeDir STARindex   --readFilesIn test.SRR493369
_1.fastq   test.SRR493369_2.fastq      --outFileNamePrefix ./LFB_HOXA1KD_repA/   --outSAMtype BAM   Unsorted      --twop
assMode Basic
@CO	user command line: STAR --genomeDir STARindex --twopassMode Basic --runThreadN 11 --outSAMtype BAM Unsorted --ou
tFileNamePrefix ./LFB_HOXA1KD_repA/ --readFilesIn test.SRR493369_1.fastq test.SRR493369_2.fastq
SRR493369.1	99	19	3977426	255	1S100M	=	3977605	488	ACTGGATCTCCACAAGGTAGATGGGCTCCATGAGGCGTGG
CTGGGCGGTCAGCACACTGGCGTAGAGGCAGCGCCGTGCTGTGGGGATGATCTGGCCCCCT	CCCFFFFFHHHHHJJJJGIJJHIIJIJIJJIIJIJJJHJJJJJIIJDGDCDEDDDC
DDCDDDB@BDDDDDDDBBB@99@BD>CCDD<@BCCCD:3@#####	NH:i:1	HI:i:1	AS:i:199	nM:i:1
SRR493369.1	147	19	3977605	255	4M208N97M	=	3977426	-488	CGCCCTCCTTGGTGGCCCACTGGAAGCCGGCC
ACCACACTGTCCTTGATCTCGTTGAGGTACTGCACACCCTTGGTGATGTCGGTGAGGATGTTGGGGCCG	5BDDBBDDDDDDBABDDDCDDDDDDDCDDDDDCDCCCADEFFFFFHHH

To count the number of rows in the header, use the Linux comand grep (though samtools view provides the -H argument to keep only the header) 

$ samtools view -h SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | grep "^@" | wc -l
$ # same as samtools view -H SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | wc -l
28
$ samtools view -h SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | grep -v "^@" | wc -l
$ # same as samtools view SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | wc -l
9856
$ samtools view -h SeqTestdata/RNASeqFibroblast/LFB_HOXA1KD_repA/accepted_hits.bam | wc -l
9884

We can also use Bioconductor's Rsamtools package to manipulate sam/bam, fasta, bcf and tabix files. 

source("https://bioconductor.org/biocLite.R")
biocLite("Rsamtools")
library(Rsamtools)
bf1 = BamFileList(file = c("file1.bam", "file2.bam"))
seqinfo(bf1)

# Suppose we want to get the MAPQ value from reads
param<-ScanBamParam(what="mapq") # other choices, what = c("rname", "strand", "pos", "qwidth", "seq")
                                 # see scanBamwhat() 
bam<-scanBam(file="filename.bam", param=param)
str(bam)
summary(bam)
# List of 1
#  $ :List of 1
#   ..$ mapq: int [1:195875920] 0 13 0 0 2 0 0 0 9 0 ...
object.size(bam)/2^20
# 747.207664489746 bytes
summary(bam[[1]][[1]])
#    Min. 1st Qu.  Median    Mean 3rd Qu.    Max.    NA's
#     0.0    60.0    60.0    58.4    60.0    60.0 2434291
table(bam[[1]][[1]])

        0         1         2         3         4         5         6         7 
  3186501     59642     40093     91541     52501     43220    123651     81155 
        8         9        10        11        12        13        14        15 
    23354    133441     19302     17924     61882     30326     17755     54093 
       16        17        18        19        20        21        22        23 
    24826     21290     52880     37874     15116     69845     25458     30183 
       24        25        26        27        28        29        30        31 
    57684     80430     13746    537584     18437     14125     41978     25748 
       32        33        34        35        36        37        38        39 
    11848     63802     13745     16987     33168     29840     12198     64799 
       40        41        42        43        44        45        46        47 
   763253     32690     61987     46020     64149     85350    129188    100040 
       48        49        50        51        52        53        54        55 
   193770    683051     64096     95453     58853     87942     58469     48584 
       56        57        58        59        60 
    50132    135261     70818    101295 185161256

Flag

https://broadinstitute.github.io/picard/explain-flags.html Interpret FLAG value (interactive)

For single end data, no flag is set so the flag is always 0.

Extract by read names with grep

# Extract reads SRR2923335.1 and SRR2923335.1999 and output only the read name (1st) and mapping quality columns (5th)
# -e = --extended-regexp, -w = matching the whole word
# awk print $1,$5 will output with a space character between columns
samtools view accepted_hits.bam  | grep -E -w "SRR2923335.1|SRR2923335.1999" | awk '{print $1,$5}'

Extract by a region

# https://www.biostars.org/p/48719/
# the output will be all reads starting anywhere between 18000-45500.
samtools view input.bam "Chr10:18000-45500" > output.bam

# https://www.biostars.org/p/45936/
# index first!!!
samtools view -h reads.bam 1:1042000-1042010 > subset.sam
samtools view -h -b reads.bam chr1:10420000-10421000 > subset.bam

chromosomes

We can use the shell command in this post to extract the chromosome name, start position from the bam file. There we can see the difference of the chromosome name of bam files created from using Ensembl and UCSC. 

# Ensembl GRCh37
brb@T3600 ~/SeqTestdata/RNASeqFibroblast $ samtools view LFB_scramble_repA/accepted_hits.bam | head | awk '{print $3}'
1
1
1
1
1

# NCBI GRCh38
brb@T3600 ~/SeqTestdata/RNASeqFibroblast $ samtools view RNAseq_Bam_ncbigrch38/LFB_scramble_repA.bam | head | awk '{print $3}'
chr1
chr1
chr1
chr1
chr1
chr1

# UCSC hg38
brb@T3600 ~/SeqTestdata/RNASeqFibroblast $ samtools view RNAseq_Bam_hg38/LFB_scramble_repA.bam | head | awk '{print $3}'
chr1
chr1
chr1
chr1
chr1
chr1

Subset reads from specific chromosomes

https://www.biostars.org/p/128967/

For example to remove mitochondrial reads from BAM files 

samtools view -b input.bam chr1 chr2 chr3 chr4 > output.bam
# Or if the bam file has been indexed (i.e. .bai file is available)
samtools idxstats input.bam | cut -f 1 | grep -v MT | xargs samtools view -b input.bam > output.bam

where samtools idxstat returns 4 columns (TAB-delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads). 

$ samtools idxstats input.bam | head
1	249250621	0	0
2	243199373	0	0
3	198022430	0	0
4	191154276	0	0
5	180915260	0	0
6	171115067	0	0
7	159138663	0	0
8	146364022	0	0
9	141213431	0	0
10	135534747	0	0

Count the number of reads with certain number of mismatches

Other alignment methods

Online tools (global and local alignments)

https://www.ebi.ac.uk/Tools/psa/

STAR

Kallisto

Subread

See Subread.

Hisat

Command line usages

Aligner Command Line
bowtie2 bowtie2 -x /path/to/bowtie2/BASENAME -1 A_1.fq,B_1.fq -2 A_2.fq,B_2.fq -S alignment.sam
tophat2 tophat2 --no-coverage-search -p $SLURM_CPUS_PER_TASK \ 
   -o OutputDir \
   /path/to/bowtie2/BASENAME \
   R1.FASTQ.gz R2.FASTQ.gz
STAR mkdir STARindex

STAR --runMode genomeGenerate \ 

  --runThreadsN $SLURM_CPUS_PER_TASK \
  --genomeDir STARindex \
  --genomeFastaFiles /path/to/fasta.fa \
  --sjdbGTFfile /path/to/genes.gtf \
  --sdjbOverhang 85

STAR --twopassMode Basic \ 

  --runThreadN $SLURM_CPUS_PER_TASK \
  --genomeDir STARindex \
  --outSAMmapqUnique 50 \
  --sjdbOverhang 85 \
  --readFilesIn r1.fastq.gz r2.fastq.gz \
  --readFilesCommand zcat \
  --outSAMtype BAM Unsorted \
  --outFileNamePrefix output/star
Subread subread-buildindex -o INDEX /path/to/fasta.fa

subread-align -T $SLURM_CPUS_PER_TASK -i INDEX -r R1.FASTQ.gz -R R2.FASTQ.gz -o output/alignment.sam

Hisat2 hisat2-build -p $SLURM_CPUS_PER_TASK \ 
 /path/to/fasta.fa INDEX

hisat2 -p $SLURM_CPUS_PER_TASK -x INDEX \ 

  -1 R1.FASTQ.gz \
  -2 R2.FASTQ.gz -S output/alignment.sam

What's special with rna-seq count data

Overdispersion

Normalization

Power Calculation

Phenotype Prediction

Improving the value of public RNA-seq expression data by phenotype prediction

Clustering

ICAclust – Independent Component Analysis (ICA) based-clustering of temporal RNA-seq data

Correlation between RNA-Seq and microarrays

Correlation between RNA-Seq and microarrays results using TCGA data

Techniques for characterizing RNA transcripts

qRT-PCR -> Microarray -> Nanostring -> RNA-Seq

Integrating RNA sequencing into neuro-oncology practice

Differential expression analysis

Reproducible RNA-Seq analysis

Other RNA-Seq data

GEO

Go to http://www.ncbi.nlm.nih.gov/sites/entrez and select 'GEO Datasets' from the drop down menu, and use 'RNA-Seq' as your search term. http://www.ncbi.nlm.nih.gov/gds?term=rna-seq

GSE28666

as used in DAFS paper. The timing is

  • bowtie2-build 115 minutes
  • fastq-dump 25 minutes per fastq (3.3 - 5.9GB fastq files)
  • tophat2 90 minutes per fastq
  • samtools 5 minutes
  • htseq-count 13 minutes

GSE30567

Used in STAR paper

GSE38886

Used in STAR paper

GSE48215 & GSE48216

Modeling precision treatment of breast cancer

GSE48215 is Whole exome Seq and GSE48216 is RNA-Seq

GSE51403 (full R script)

RNA-seq differential expression studies: more sequence, or more replication?

An example of R code to download SRA files. 

        homo sapiens,
        Illumina HiSeq 2000.
        Question: what reference genome I should use?
        Ans1: hg18 was used.
              See http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1244821
              Or the paper.

        Ans2: GRCh38:
              Click 'BioProject' in http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51403
                then click 'Genome' in http://www.ncbi.nlm.nih.gov/bioproject/PRJNA222975
                But tophat website only provides GRCh37.
                https://genome.ucsc.edu/goldenPath/newsarch.html shows GRCh38 assembly is released
                in Dec/2013.

        38 fastq/SRR  (all are single lane)
        14 samples/GSM

        GSM#    SRX#    Sample          SRR#
       ---------------------------------------------------------------
Ctrl    1244822 365217  Ctrl_Rep7       1012952, 1012953, 1012954 (3 runs, 70M spots, 3.5G bases)
        1244821 365216  Ctrl_Rep6       949-951 (I just skip prefix '1012')
        1244820 365215  Ctrl_Rep5       946-948 (3 runs)
        1244819 365214  Ctrl_Rep4       943-945 (3 runs)  Control ethanol 24h
        1244818 365213  Ctrl_Rep3       940-942 (3 runs)
        1244817 365212  Ctrl_Rep2       937-939 (3 runs)
        1244816 365211  Ctrl_Rep1       934-936 (3 runs)  Control ethanol 24h

Trmnt   1244815 365210  E2_Rep7         931-933 (3 runs)  10nM E2 treatment for 24h
        1244814 365209  E2_Rep6         928-930 (3 runs)
        1244813 365208  E2_Rep5         925-927 (3 runs)
        1244812 365207  E2_Rep4         923-924 (2 runs)
        1244811 365206  E2_Rep3         921-922 (2 runs)
        1244810 365205  E2_Rep2         919-920 (2 runs)  10nM E2 treatment for 24h
        1244809 365204  E2_Rep1         917-918 (2 runs)  10nM E2 treatment for 24h

# Step 1. Download and convert data

library(SRAdb)
setwd("~/GSE51403")
 if( ! file.exists('SRAmetadb.sqlite') ) {
        # sqlfile <- getSRAdbFile()
   sqlfile <- "~/Anders2013/SRAmetadb.sqlite"
 } else {
sqlfile <- 'SRAmetadb.sqlite'
 }
sra_con <- dbConnect(SQLite(),sqlfile)
fs <- listSRAfile("SRP031476", sra_con, fileType = "sra" )     # 38 files
# getSRAfile("SRP031476", sra_con, fileType = "sra" )          # starting to download

getSRAfile(fs$run[13], sra_con, fileType='sra') # SRR1012917.sra
getSRAfile(fs$run[30], sra_con, fileType='sra') # SRR1012918.sra

dbDisconnect( sra_con )

fs <- dir(pattern='sra')
# for(f in fs)  system(paste("fastq-dump --split-3", f)) # Single thread, Not efficient
scrp <- paste("/opt/RNA-Seq/bin/sratoolkit.2.3.5-2-ubuntu64/bin/fastq-dump --split-3", fs)

library(parallel)
cl <- makeCluster(getOption("cl.cores", 5))
# note that if something is wrong, we need to delete broken fastq first and then run again.
system.time(clusterApply(cl, scrp, function(x) system(x))) 
stopCluster(cl)

# Step 2. Create samples object
samples <- data.frame(LibraryName = c(paste("Ctrl_Rep", 7:1, sep=''),
                                      paste("E2_Rep", 7:1, sep='')),
             LibraryLayout = rep("SINGLE", 14),
             fastq1=c(paste(paste("SRR", 1012952:1012954, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012949:1012951, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012946:1012948, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012943:1012945, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012940:1012942, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012937:1012939, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012934:1012936, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012931:1012933, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012928:1012930, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012925:1012927, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012923:1012924, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012921:1012922, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012919:1012920, ".fastq", sep=''), collapse=','),
                      paste(paste("SRR", 1012917:1012918, ".fastq", sep=''), collapse=',')),
             fastq2=rep("", 14),
             countf=c(paste("Ctrl_Rep", 7:1, ".count", sep=''),
                      paste("E2_Rep", 7:1, ".count", sep='')))
# Step 3: run tophat2
i <- 10
system.time(paste("tophat2 -G genes.gtf -p 5 -o",
                   samples$LibraryName[i],
                   "genome",
                   samples$fastq1[i]))
# system("tophat2 -G genes.gtf -p 5 -o E2_Rep5 genome SRR1012925.fastq")
# system("tophat2 -G genes.gtf -p 5 -o E2_Rep5 genome SRR1012925.fastq,SRR1012926.fastq,SRR1012927.fastq") 
for(i in c(1:14)) {
  system(paste("tophat2 -G genes.gtf -p 5 -o", samples$LibraryName[i], "genome", samples$fastq1[i]))
}

# Step 4. Organize, sort and index the BAM files and create SAM files.
for(i in seq_len(nrow(samples)) {
  lib = samples$LibraryName[i]
  ob = file.path(lib, "accepted_hits.bam")
  # sort by name, convert to SAM for htseq-count
  c1 <- paste0("samtools sort -n ", ob, " ", lib, "_sn")
  system(c1)
}

samtools sort -n Sample1/accepted_hits.bam Sample1_sn  # 283 seconds
# samtools sort Sample1/accepted_hits.bam Sample1_s

# Step 5. Count reads using htseq-count
htseq-count -f bam -s no -a 10 Sample1_sn.bam Mus_musculus.GRCm38.70.gtf > Sample1.count # 759 seconds
htseq-count -f bam -s no -a 10 Sample2_sn.bam Mus_musculus.GRCm38.70.gtf > Sample2.count
samples$countsf <- paste(samples$LibraryName, "count", sep=".")
gf <- "genes.gtf"
cmd <- paste0("htseq-count -s no -a 10 ", samples$LibraryName, "_sn.sam ", gf, " > ", samples$countf)
system.time(clusterApply(cl, cmd, function(x) system(x))) # 119 minutes


samplesDESeq = data.frame(shortname = c("Sample1", "Sample2"), countf=c("Sample1.count", "Sample2.count"),
                         condition = c("CTL", "KD"), LibraryLayout =c("SINGLE", "SINGLE"))
library("DESeq")
cds = newCountDataSetFromHTSeqCount(samplesDESeq)
cds = estimateSizeFactors(cds)
sizeFactors(cds)

GSE37544

RNA-seq analysis of differential gene expression in liver from lactating dairy cows divergent in negative energy balance

GSE11045

Probe Region Expression Estimation for RNA-Seq Data for Improved Microarray Comparability

GSE37703/SRP012607

Differential analysis of HOXA1 in adult cells at isoform resolution by RNA-Seq by Trapnell C, Hendrickson DG, Sauvageau M, Goff L et al. Nat Biotechnol 2013.

The <samples.txt> file required by BRB-SeqTools looks like 

LibraryName	LibraryLayout	fastq1	fastq2	ReadGroup	SequencerManufacturer
LFB_scramble_repA	PAIRED	SRR493366_1.fastq	SRR493366_2.fastq	1	illumina
LFB_scramble_repB	PAIRED	SRR493367_1.fastq	SRR493367_2.fastq	2	illumina
LFB_scramble_repC	PAIRED	SRR493368_1.fastq	SRR493368_2.fastq	3	illumina
LFB_HOXA1KD_repA	PAIRED	SRR493369_1.fastq	SRR493369_2.fastq	4	illumina
LFB_HOXA1KD_repB	PAIRED	SRR493370_1.fastq	SRR493370_2.fastq	5	illumina
LFB_HOXA1KD_repC	PAIRED	SRR493371_1.fastq	SRR493371_2.fastq	6	illumina

GSE37704

http://www.gettinggeneticsdone.com/2015/12/tutorial-rna-seq-differential.html

GSE64570, GSE69244, GSE72165

Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences

Ion Torrent

  • GEO
  • GSE46876 from GPL17301 PGM (Homo sapiens) and GPL17303 Ion Torrent Proton (Homo sapiens). Unequal read length.
  • GSE83668 from GPL17303 Ion Torrent Proton (Homo sapiens) and GPL11154 Illumina HiSeq 2000 (Homo sapiens)
  • GSE79051/SRP071553 from GPL17303 Ion Torrent Proton (unequal read length)

SRA

Pipeline

Extracting allelic read counts from 250,000 human sequencing runs in Sequence Read Archive 2018. Code in Github and Data.

SRA000299

Note that one fastq file can represent several channels/lanes. The lane information can be extracted from fastq definition line (first line of each read). For example, 

@SRR002321.1 080226_CMLIVERKIDNEY_0007:2:1:115:885

"SRR002321.1" is the short read archive name, where the .1 is the read number, "080226_CMLIVERKIDNEY_0007" should be the Machine name, which has been arbitrarily changed

  • "2" is the Channel/lane number
  • "1" is the Tile number
  • "115" is the X position
  • "885" is the Y position

The website also shows how to extract reads with the same run from fastq files.

In this example, there are

  • two full sequencing runs (this info can be obtained from paper)
  • 4 experiments (SRX000571, SRX000604, SRX000605, SRX000606)
  • 6 runs (SRRR002321, SRR002323, SRR002322, SRR002320, SRR002325, SRR002324)
  • 7 lanes (eg "080226_CMLIVERKIDNEY_0007:2") for each full sequencing run

SRA data linked from QuickNGS

Include Chip-Seq, RNA-Seq, WGS and miRNA-Seq. QuickNGS is a production environment for quick batch-wise analysis of Next-Generation Sequencing (NGS) data in high-throughput laboratories.

European Nucleotide Archive/ENA

DDBJ

ENCODE

http://www.ncbi.nlm.nih.gov/geo/info/ENCODE.html has a list of data with GSE number.

Data Processing Pipelines

1000 genomes

Human BodyMap 2.0 data from Illumina

TCGA/The Cancer Genome Atlas

> if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
> BiocManager::install("RTCGA.clinical")
> library(RTCGA.clinical)

# Available cohorts
> checkTCGA('Dates')
 [1] "2011-10-26" "2011-11-15" "2011-11-28" "2011-12-06" "2011-12-30"
 [6] "2012-01-10" "2012-01-24" "2012-02-17" "2012-03-06" "2012-03-21"
[11] "2012-04-12" "2012-04-25" "2012-05-15" "2012-05-25" "2012-06-06"
[16] "2012-06-23" "2012-07-07" "2012-07-25" "2012-08-04" "2012-08-25"
[21] "2012-09-13" "2012-10-04" "2012-10-18" "2012-10-20" "2012-10-24"
[26] "2012-11-02" "2012-11-14" "2012-12-06" "2012-12-21" "2013-01-16"
[31] "2013-02-03" "2013-02-22" "2013-03-09" "2013-03-26" "2013-04-06"
[36] "2013-04-21" "2013-05-08" "2013-05-23" "2013-06-06" "2013-06-23"
[41] "2013-07-15" "2013-08-09" "2013-09-23" "2013-10-10" "2013-11-14"
[46] "2013-12-10" "2014-01-15" "2014-02-15" "2014-03-16" "2014-04-16"
[51] "2014-05-18" "2014-06-14" "2014-07-15" "2014-09-02" "2014-10-17"
[56] "2014-12-06" "2015-02-02" "2015-02-04" "2015-04-02" "2015-06-01"
[61] "2015-08-21" "2015-11-01" "2016-01-28"
# Available cohorts
> (cohorts <- infoTCGA() %>% rownames() %>% sub("-counts", "", x=.))

 [1] "ACC"      "BLCA"     "BRCA"     "CESC"     "CHOL"     "COAD"    
 [7] "COADREAD" "DLBC"     "ESCA"     "FPPP"     "GBM"      "GBMLGG"  
[13] "HNSC"     "KICH"     "KIPAN"    "KIRC"     "KIRP"     "LAML"    
[19] "LGG"      "LIHC"     "LUAD"     "LUSC"     "MESO"     "OV"      
[25] "PAAD"     "PCPG"     "PRAD"     "READ"     "SARC"     "SKCM"    
[31] "STAD"     "STES"     "TGCT"     "THCA"     "THYM"     "UCEC"    
[37] "UCS"      "UVM" 
> dim(BRCA.clinical)
[1] 1098 3703
> dim(OV.clinical) # one row per sample
[1]  591 3626
> dim(LUAD.clinical)
[1]  522 3046
> OV.survInfo <- survivalTCGA(OV.clinical, extract.cols = "admin.disease_code") 
> dim(OV.survInfo)  # 576 x 4
> plot(survfit(Surv(times, patient.vital_status) ~ 1, 
       data = OV.survInfo), conf.int=F, mark.time=TRUE)

> BiocManager::install("RTCGA.mRNA")
> data(package = "RTCGA.mRNA")
> data(OV.mRNA, package = "RTCGA.mRNA")
> dim(OV.mRNA)  # one row per sample
[1]   561 17815
> OV.mRNA[1:2, 1:5]
           bcr_patient_barcode       ELMO2  CREB3L1    RPS11   PNMA1
1 TCGA-01-0628-11A-01R-0363-07 -0.24800000 -0.14150 1.370625 1.08775
2 TCGA-01-0639-11A-01R-0363-07  0.09508333 -0.04650 0.982875 1.38725
> OV.clinical[1:2, 18]
[1] "tcga-04-1331" "tcga-04-1332"

> match(substr(OV.mRNA[, 1], 6, 12), substr(OV.clinical[, 18], 6, 12)) %>% 
  na.omit() %>% length()
[1] 546

The TCGA Data Portal does not host lower levels of sequence data. NCI's Cancer Genomics Hub (CGHub) is the new secure repository for storing, cataloging, and accessing BAM files and metadata for sequencing data (need to 1. register from HHS to get an access 2. request access to the secure data set).

UCI

ReCount

Other RNA-Seq tools

http://en.wikipedia.org/wiki/List_of_RNA-Seq_bioinformatics_tools

Galaxy

The first 3 lines will install galaxy on local computer. The last command will start Galaxy. PS. It will take a while to start the first time. 

hg clone https://bitbucket.org/galaxy/galaxy-dist/
cd galaxy-dist
hg update stable
sh run.sh

A screenshot of the available tools is given below. Strangely, I did not find "NGS: RNA Analysis" in the NGS: ToolBox on left hand side.

GalaxyInitial.png

We cannot upload > 2GB fastq data. For such large dataset (we have one 3.3 GB fastq in this case), Galaxy asks to use http or ftp to load the data. I install Apache in Ubuntu and put this fastq file there for upload.

Galaxy does not include bowtie, tophat, .... by default. See this post and Galaxy wiki about the installation. Another option is to modify line 614 of 'universe_wsgl.ini' file and enable ourselves as adminstrator. Also modify line 146 to add a path for installing tools through Galaxy Admin page.

If you submit a lot of jobs (eg each tophat2 generates 5 jobs) some of jobs cannot be run immediately. In my experience, there is a 25 jobs cap. Running jobs has a yellow background color. Waiting for run jobs has a gray background color.

Some helpful information

Cufflink -> Cuffcompare or Cuffmerge -> Cuffdiff

Forum

recount2

A multi-experiment resource of analysis-ready RNA-seq gene and exon count datasets https://jhubiostatistics.shinyapps.io/recount/

recount3

RNACocktail

A comprehensive framework for accurate and efficient RNA-Seq analysis

Massive Mining of Publicly Available RNA-seq Data from Human and Mouse

Misc

Markets

Global And United States NGS-based RNA-seq Market 2017

Tutorial/Primer

Whole Transcriptome Profiling: An RNA-Seq Primer and Implications for Pharmacogenomics Research

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