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.
- 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).
- 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.
- 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.
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 |
|---|---|---|
| bowtie2 | C++ | 2.2.6 |
| tophat | C++ | 2.1.0 |
| bwa | C | 0.7.12 |
| star | C++ | 2.5.1b |
| picard | Java | 1.141 |
| samtools | C | 1.3 |
| GATK* | Java | 3.6 |
| bcftools | C | 1.3 |
| htslib | C | 1.3 |
| annovar* | Perl | 2016Feb01 |
| sratoolkit | Shell | 2.7.0 |
| fastqc | Java | 0.11.5 |
| fastx | C | 0.0.13 |
| snpeff | Java | 4_2 |
| htseq | Python | 0.6.1p1 |
| R | R | 3.3.x |
| pandoc | Haskell | 1.16.0.2 |
| latex, lftp, avfs | Ubuntu repository |
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 200GB dynamic allocated space VM,
| space in GB | total | used | avail | vdi | ova |
|---|---|---|---|---|---|
| After Ubuntu installation | 189 | 3.7 | 176 | 4.5 | 1.7 |
| After running Automatic setup (Tools Manager) | 189 | 6.9 | 172 | 8.4 | 4.2 |
| After running the RNS-Seq sample data | 189 | ||||
| After running the DNS-Seq sample data | 189 | ||||
| After running snpEff on the DNS-Seq sample data | 189 |
Tips
Tutorial videos
https://www.youtube.com/playlist?list=PL6A4OqNJzh1l1CnCRdO_Q7o_-0K5CjrP4