Tassel 5 GBS v2 Pipeline sample script for calling SNPs using reference genome

For this script to work properly, you should have gbs folder in your home directory, and jobs and projects folders inside gbs

Only copy the code, not the guiding text. Once you have the full script copied in a simple text file with extension .sh, the name variable should be replaced with something more explicit, such as hessian_F3 for a Hessian fly project with F3 population. To do this in a semi-automatic fashion, use find and replace function in your text editor and replace name variable with hessian_F3 or any other project name.

To keep everything organized, I recommend using the same name for the project and the files, for example

Project name - hessian_F3

Keyfile - hessian_F3.txt

Shell script - hessian_F3.sh

Script can be also downloaded from here, but make sure to copy and paste in a simple text file with .sh extension, for example hessian_F3.sh.

Once the Keyfile and shell script are ready, put them in jobs folder and run qsub hessian_F3.sh

• Start your script with the following piece of code. First line is called Shebang and is required for a shell script. The PATH variable can be changed if the software is installed elsewhere, otherwise, leave it as it is

#!/bin/bash
export PATH=$PATH:/homes/nss470/usr/bin:/homes/nss470/usr/bin/bin

• Update the user variable with your K-State eID

user=eID

• Requesting beocat resources

#$ -l h_rt=60:00:00
#$ -l mem=64G
#$ -M your_email -m aes
#$ -N name
#$ -cwd

• Your keyfile should have following four mandatory headers

'Flowcell' 'Lane' 'Barcode' 'FullSampleName'

keyFile=/homes/$user/gbs/jobs/name.txt

• Path to sequence directory

seqDir=/bulk/jpoland/sequence

• Database path (reference genome)

dbPath=/bulk/jpoland/genome/ChineseSpring/pseudomolecules_v1.0/161010_Chinese_Spring_v1.0_pseudomolecules_parts

• TASSEL5 path

tasselPath=/homes/nss470/softwares/tassel5/run_pipeline.pl

• Create required directories for output

mkdir /homes/$user/gbs/projects/name
mkdir /homes/$user/gbs/projects/name/keyFileSh
cd /homes/$user/gbs/projects/name

• Running TASSEL 5 SNP calling pipeline in series of steps. Each step uses mostly the default settings that can be changed if desired

## GBSSeqToTagDBPlugin - RUN Tags to DB
$tasselPath -Xms64G -Xmx64G -fork1 -GBSSeqToTagDBPlugin -e PstI-MspI \
    -i $seqDir \
    -db name.db \
    -k $keyFile \
    -kmerLength 64 -minKmerL 20 -mnQS 20 -mxKmerNum 250000000 \
    -endPlugin -runfork1 >> z_name_GBSSeqToTagDB.out
 
## TagExportToFastqPlugin - export Tags
$tasselPath -fork1 -TagExportToFastqPlugin \
    -db name.db \
    -o name_tagsForAlign.fq -c 1 \
    -endPlugin -runfork1 >> z_name_TagExportToFastq.out
 
## RUN BOWTIE
bowtie2 -p 15 --very-sensitive-local \
    -x $dbPath \
    -U name_tagsForAlign.fq \
    -S name.sam >> z_name_bowtie2.out
 
## SAMToGBSdbPlugin - SAM to DB
$tasselPath -Xms64G -Xmx64G -fork1 -SAMToGBSdbPlugin \
    -i name.sam \
    -db name.db \
    -aProp 0.0 -aLen 0 \
    -endPlugin -runfork1 >> z_name_SAMToGBSdb.out
 
## DiscoverySNPCallerPluginV2 - RUN DISCOVERY SNP CALLER
$tasselPath -Xms64G -Xmx64G -fork1 -DiscoverySNPCallerPluginV2 \
    -db name.db \
    -mnLCov 0.1 -mnMAF 0.01 -deleteOldData true \
    -endPlugin -runfork1 >> z_name_DiscoverySNPCaller.out
 
## SNPQualityProfilerPlugin - RUN QUALITY PROFILER
$tasselPath -Xms64G -Xmx64G -fork1 -SNPQualityProfilerPlugin \
    -db name.db \
    -statFile name_SNPqual_stats.txt \
    -endPlugin -runfork1 >> z_name_SNPQualityProfiler.out
 
## UpdateSNPPositionQualityPlugin - UPDATE DATABASE WITH QUALITY SCORE
$tasselPath -Xms64G -Xmx64G -fork1 -UpdateSNPPositionQualityPlugin \
    -db name.db \
    -qsFile name_SNPqual_stats.txt \
    -endPlugin -runfork1 >> z_name_UpdateSNPPositionQuality.out
 
## ProductionSNPCallerPluginV2 - RUN PRODUCTION PIPELINE - output .h5
$tasselPath -Xms64G -Xmx64G -fork1 -ProductionSNPCallerPluginV2 \
    -db name.db \
    -i $seqDir \
    -k $keyFile \
    -o name.h5 \
    -e PstI-MspI -kmerLength 64 \
    -endPlugin -runfork1 >> z_name_ProductionSNPCaller.out
 
## ProductionSNPCallerPluginV2 - RUN PRODUCTION PIPELINE - output .vcf
$tasselPath -Xms64G -Xmx64G -fork1 -ProductionSNPCallerPluginV2 \
    -db name.db \
    -i $seqDir \
    -k $keyFile \
    -o name.vcf \
    -e PstI-MspI -kmerLength 64 \
    -endPlugin -runfork1 >> z_name_ProductionSNPCaller.out
 
## Convert to Hapmap format
$tasselPath -Xms64G -Xmx64G -fork1 -vcf name.vcf \
	-export name -exportType Hapmap

• Copy keyfile and .sh file for backup

mv /homes/$user/gbs/jobs/name.* keyFileSh/

If everything ran correctly, you should get a folder named hessian_F3 in your gbs/projects folder with all the output files including SNPs containing .vcf and .hmp.txt files that can be used for further analyses