View source for 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' ====

Once you have the full script copied in a 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.

<pre>
Script can be downloaded from [[Media:Tassel5v2_sample_script.docx|here]] too, but make sure to copy and paste in a simple text file with '.sh' extension, for example '''hessian_F3.sh'''
</pre>

''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''

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


''Update the ''<code>user</code> ''variable with your K-State eID''
<pre>
user=eID
</pre>


''Requesting beocat resources''
<pre>
#$ -l h_rt=60:00:00
#$ -l mem=64G
#$ -M your_email -m aes
#$ -N name
#$ -cwd
</pre>


''Path to keyfile --> '''keyfile''' should have following 4 mandatory headers''

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

''Place the keyfile in your 'jobs' folder. It should be renamed to the what you are replacing the name variable with, for example 'hessian_F3.txt'''
<pre>
keyFile=/homes/$user/gbs/jobs/name.txt
</pre>


''Path to sequence directory''
<pre>
seqDir=/bulk/jpoland/sequence
</pre>


''Database path (reference genome)''
<pre>
dbPath=/bulk/jpoland/genome/ChineseSpring/pseudomolecules_v1.0/161010_Chinese_Spring_v1.0_pseudomolecules_parts
</pre>


''TASSEL5 path''
<pre>
tasselPath=/homes/nss470/softwares/tassel5/run_pipeline.pl
</pre>


''Create required directories for output''
<pre>
mkdir /homes/$user/gbs/projects/name
mkdir /homes/$user/gbs/projects/name/keyFileSh
cd /homes/$user/gbs/projects/name
</pre>


''Running TASSEL 5 SNP calling pipeline in series of steps. Each step uses mostly the default settings that can be changed if desired''
<syntaxhighlight lang="java">
## 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
</syntaxhighlight>


''Copy keyfile and .sh file for backup''
<pre>
mv /homes/$user/gbs/jobs/name.* keyFileSh/
</pre>


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

==== End ====