Seqtools

Wiki for BRB-SeqTools

Windows 10 Bash shell

Gene counting and variant call (both Samtools and GATK) works fine. For variant annotation, see the comment #2 below.

1. Need to install Xming. Before calling ./SeqTools from the Bash shell, run export DISPLAY=:0 first.
   • If we like to start Xming automatically when Windows boots, follow the instruction How to Make a Program Run at Startup on Any Computer.
   • Press Windows + R (or 'run' in the search box) and click the Enter key. Type “shell:startup” into the Run dialog, and press Enter. Now drag-and-drop the Xming shortcut from the “All Apps” list in the Start menu directly into this folder. Reboot Windows to make this change to work. It also helps to download the BRB-SeqTools icon (read below).
2. Automatic setup:
   • Need to install unzip utility sudo apt-get install unzip.
   • Install fontconfig library sudo apt-get install libfontconfig1-dev.
   • New gnome-terminal windows (use apt-get install) cannot be opened from Bash shell. This affect Automatic setup tools in Tools manager and profile manager.
   • Java JDK from ppa:webup8team does not work. We need to download/install it <jdk-8u112-linux-x64.tar.gz> from Oracle website. To make the installation silently, we need to add two lines to the installation script. See here for apt-get approach and here for tarball approach.
   • An issue in pandoc: timer-create function not implemented. In other words, if we run variant annotation, we will get a bug report message. The main output files (1 vcf and 2 texts files) are generated but the pdf/html files cannot be created.
3. It is useful to create a Windows icon on the Windows desktop for quick access to BRB-SeqTools program. The BRB-SeqTools icon <BRB-SeqTools.lnk> can be found on Github. A modified automatic setup script <install_rnaseq.sh> can be also found there.

Performance

A subset of GSE48215 (about 1/10 of the original FASTQ files) created to run the benchmark.

mkdir GSE48215_22000000
head -n 22000000 GSE48215/SRR925751_1.fastq  > GSE48215_22000000/SRR925751_1.fastq
head -n 22000000 GSE48215/SRR925751_2.fastq  > GSE48215_22000000/SRR925751_2.fastq

The reference genome file is based on UCSC_hg19_chr1 as part of the DNA-Seq sample data.

	time (min)
Ubuntu 14.04 host	11
Ubuntu 16.04 vm	26
Windows 10 vm	32

Both virtual machines have 6 cores CPU and 16GB memory. For this dataset, about 8GB memory is enough. VirtualBox 5.0.30 was used.

Software List

See Tools Manager -> Automatic setup. A developer version of the shell script is available on Github. Note: GATK and annovar will not be installed automatically due to the license issue.

Program	Major language	Version	Linux OS	Mac OS	Repository
bowtie2	C++	2.2.6	src & binary zip	src & binary zip	Github & SourceForge
tophat	C++	2.1.0	Linux binary tar.gz	Mac binary tar.gz	jhu.edu
bwa	C	0.7.12	src	src	Github
star	C++	2.5.1b	one binary tar.gz	one binary tar.gz	Github
picard	Java	1.141			Github
samtools	C	1.3	src	src	Github
GATK*	Java	3.6			broadinstitute.org
bcftools	C	1.3	src	src	Github
htslib	C	1.3	src	src	Github
annovar*	Perl	2016Feb01			openbioinformatics.org
sratoolkit	Shell	2.7.0	Linux binary tar.gz	Mac binary tar.gz	nih.gov & Github
fastqc	Java	0.11.5		dmg	bioinformatics.babraham.ac.uk
fastx	C	0.0.13	Linux binary tar.bz2	Mac binary tar.bz2	hannonlab.cshl.edu
snpeff	Java	4_2			SourceForge & Github
htseq	Python	0.6.1p1	src	src	python.org
R	R	3.3.x	apt-get	app	cran.rstudio.com
pandoc	Haskell	1.16.0.2	deb	pkg	Github
latex		Ubuntu repository	apt-get	pkg	Ubuntu/tug.org
lftp		Ubuntu repository	apt-get	homebrew	Ubuntu/Rudix
avfs		Ubuntu repository	apt-get	src	Ubuntu/SourceForge
Java (jdk)		1.8.0 (8u112)	tar.gz	dmg	oracle.com
subread	C	1.5.2	src & binary	src & binary	SourceForge

Download failure

Several software repositories (eg Github, not sourceforge) are hosted by Amazon S3. So be ware of possible Amazon AWS outage.

• Amazon AWS status
• Github status

Hard Disk Space

• Tools Manager: Automatic setup will download 1.2GB data and take about 3GB disk space. Automatic setup will download tools required for automatic method in Profile Manager and COSMIC data download in Variant Annotation.
• Profile Manager: Most genomes except hg19 will download 20GB data (hg19 will download 40GB data).
• Variant Annotation: Each of snpEff and ANNOVAR will download 15GB database for dbNSFP.

Virtual machine

For a 100GB dynamic allocated space VM,

space in GB	total	used	avail	vdi	ova
After Ubuntu installation	89	3.7	76	4.5	1.7
After running Automatic setup (Tools Manager)	89	6.9	72	8.4	4.2
After running the RNS-Seq sample data	89
After running the DNS-Seq sample data	89
After running snpEff on the DNS-Seq sample data	89

Predefined Locations

• Demo data: testdata (parent directory is determined by the user)
• Reference genome from the automatic method: BRB_SeqTools_autosetup_reference_genome_files (parent directory is determined by the user)
• Database files from somatic mutation annotator tool: ~/variantAnnoDatabase

Reference sequencing and annotation files from iGenomes

Note that the file size may change as the file is updated.

	size (bytes)	md5sum
Ensembl GRCh37 Homo_sapiens_Ensembl_GRCh37.tar.gz	19971514224	3077c2b593615e418160ae878009b4b5
NCBI GRCh38 Homo_sapiens_NCBI_GRCh38.tar.gz	15848139211	61d263698f0283075f63b1514a16045d
UCSC hg19 Homo_sapiens_UCSC_hg19.tar.gz	45468620403	b64a925c6b7cc0391b3ee188ecec4e48
UCSC hg38 Homo_sapiens_UCSC_hg38.tar.gz	16006984068	97d98dcb25fb041be2f2139322a84cdb

Note that the download speed is about 3MB/s at home and 7.8~11.4MB/s at office (shown at the end of stdout using wget).

Tips

Tutorial videos

https://www.youtube.com/playlist?list=PL6A4OqNJzh1l1CnCRdO_Q7o_-0K5CjrP4