ahcg_pipeline

Variant calling pipeline for genomic data analysis

Download Virtualbox

https://www.virtualbox.org/wiki/Downloads

Set up Virtualbox

Download .ova file

https://da1s119xsxmu0.cloudfront.net/sites/developer/native/nativeappsvm/BaseSpace%20Native%20App%20VM%20(phix%20only)%20v9.ova

#Connect via putty Host: vagrant@localhost Password: vagrant Port: 2222

Clone Shashi's AHCG Pipeline

git clone https://github.com/shashidhar22/ahcg_pipeline.git

Requirements: Can be downloaded manually or use git pull origin master

1. Python3 - version 3.4.1
2. Trimmomatic - version 0.36
3. Bowtie2 - version 2.2.9
4. Picard tools - version 2.6.0
5. GATK - version 3.4

Java install

Tried installing java from source, ran into many issues. Installed via sudo.

sudo apt-get install jre-default

java -jar picard.jar CreateSequenceDictionary R=hg.19.fa O=hg19.dict

Install samtools

Installed samtools (& bcftools, htslib) via source. samtools-1.3.1 bcftools-1.3.1 htslib-1.3.1

cd samtools-1.3.1 make make prefix=/home/bin install

[Edit .bashrc] PATH=$PATH:/home/bin export PATH

samtools faidx

samtools faidx hg19.fa

bowtie index

Originally downloaded bowtie index from Shashi, but those were not compatible with the reference file. So the index files were built from bowtie.

bowtie2-build -f /path/to/hg19.fa hg19

Download the reference genome

wget www.prism.gatech.edu/~sravishankar9/resources.tar.gz

gunzip resources.tar.gz

tar -xvf resources.tar

Download NIST test data

wget ftp://ftp-trace.ncbi.nih.gov/giab/ftp/data/NA12878/Garvan_NA12878_HG001_HiSeq_Exome/NIST7035_TAAGGCGA_L001_R1_001.fastq.gz

wget ftp://ftp-trace.ncbi.nih.gov/giab/ftp/data/NA12878/Garvan_NA12878_HG001_HiSeq_Exome/NIST7035_TAAGGCGA_L001_R2_001.fastq.gz

gunzip NIST7035_TAAGGCGA_L001_R1_001.fastq.gz

gunzip NIST7035_TAAGGCGA_L001_R2_001.fastq.gz

head -100000 NIST7035_TAAGGCGA_L001_R1_001.fastq > test_r1.fastq

head -100000 NIST7035_TAAGGCGA_L001_R2_001.fastq > test_r2.fastq

Run the ahcg pipeline

python3 ahcg_pipeline.py -t ./lib/Trimmomatic-0.36/trimmomatic-0.36.jar -b ./lib/bowtie2-2.2.9/bowtie2 -p ./lib/picard.jar -g ./lib/GenomeAnalysisTK.jar -i ./test_r*.fastq -w ./hg19 -d ./resources/dbsnp/dbsnp_138.hg19.vcf -r ./resources/genome/hg19.fa -a ./lib/Trimmomatic-0.36/adapters/TruSeq2-PE.fa -o outputdir

###GIT SECTION

git ssh

Added github ssh key 'biol'

changing remotes

##Move to desired directory cd ahcg_pipeline/

##List current remotes git remote -v

[origin https://github.com/shashidhar22/ahcg_pipeline.git (fetch)] [origin https://github.com/shashidhar22/ahcg_pipeline.git (push)]

##Change my remote from HTTPS to SSH git remote set-url origin git@github.com:jmoore335/ahcg_pipeline.git

##Check that the remote URL changed git remote -v

[origin git@github.com/jmoore335/ahcg_pipeline.git (fetch)] [origin git@github.com/jmoore335/ahcg_pipeline.git (push)]

git ignore

Add the directories you don't wish to commit to git ignore

vim git.ignore

Add .fastq files because of large size.

git commit instructions

git add.README.md git commit -m "revisions, should be more detailed" git config --global user.email jmoore335@gatech.edu git commit -m "revisions, should be more detailed" git push origin master

The revised README file has now be committed to github.

Reference genome

Reference genomes can be downloaded from Illumina iGenomes

Test data

Use the following protocol to download and prepare test dataset from NIST sample NA12878

wget ftp://ftp-trace.ncbi.nih.gov/giab/ftp/data/NA12878/Garvan_NA12878_HG001_HiSeq_Exome/NIST7035_TAAGGCGA_L001_R1_001.fastq.gz
wget ftp://ftp-trace.ncbi.nih.gov/giab/ftp/data/NA12878/Garvan_NA12878_HG001_HiSeq_Exome/NIST7035_TAAGGCGA_L001_R2_001.fastq.gz
gunzip NIST7035_TAAGGCGA_L001_R1_001.fastq.gz
gunzip NIST7035_TAAGGCGA_L001_R2_001.fastq.gz
head -100000 NIST7035_TAAGGCGA_L001_R1_001.fastq > test_r1.fastq
head -100000 NIST7035_TAAGGCGA_L001_R2_001.fastq > test_r2.fastq

Help

To access help use the following command:

python3 ahcg_pipeline.py -h