GBSv2 Discovery/Production Pipeline Overview 

1) GBSSeqToTagDBPlugin


./tassel-5-standalone/run_pipeline.pl -Xms100G -Xmx128G -fork1 -GBSSeqToTagDBPlugin -e EcoT22I -i Tassel5GBSv2Pipeline -db Tassel5GBSv2Pipeline/GooseGBS.db -k Tassel5GBSv2Pipeline/Goose_Keyfile_Tassel5GBSv2Pipeline.txt -kmerLength 64 -minKmerL 20 -mnQS 20 -mxKmerNum 100000000 -endPlugin -runfork1


2) TagExportToFastqPlugin


./tassel-5-standalone/run_pipeline.pl -fork1 -TagExportToFastqPlugin -db Tassel5GBSv2Pipeline/GooseGBS.db -o Tassel5GBSv2Pipeline/GooseTags.fa.gz -c 1 -endPlugin -runfork1


3) Run Alignment programs
#The sequencing data was aligned to reference genome with Burrows-Wheeler Aligner
  
bwa aln -t 4 -n 0.04 Tassel5GBSv2Pipeline/referenceSequence/Chinese_Goose_genome.fna Tassel5GBSv2Pipeline/GooseTags.fa.gz > Tassel5GBSv2Pipeline/GooseTagsForAlign.sai

bwa samse Tassel5GBSv2Pipeline/referenceSequence/Chinese_Goose_genome.fna Tassel5GBSv2Pipeline/GooseTagsForAlign.sai Tassel5GBSv2Pipeline/GooseTags.fa.gz > Tassel5GBSv2Pipeline/GooseTagsForAlign.sam



4) SAMToGBSdbPlugin
 

./tassel-5-standalone/run_pipeline.pl -fork1 -SAMToGBSdbPlugin -i Tassel5GBSv2Pipeline/GooseTagsForAlign.sam -db Tassel5GBSv2Pipeline/GooseGBS.db -endPlugin -runfork1



5) DiscoverySNPCallerPluginV2


./tassel-5-standalone/run_pipeline.pl -fork1 -DiscoverySNPCallerPluginV2 -db Tassel5GBSv2Pipeline/GooseGBS.db -sC 1 -mnLCov 0.8 -mnMAF 0.01 -deleteOldData true -endPlugin -runfork1



6) SNPQualityProfilerPlugin


./tassel-5-standalone/run_pipeline.pl -fork1 -SNPQualityProfilerPlugin -db ./Tassel5GBSv2Pipeline/GooseGBS.db -taxa ./Tassel5GBSv2Pipeline/GBSGooseTaxaNames.txt -tname "Goose" -statFile "OutputGooseStats.txt" -deleteOldData true -endPlugin -runfork1



7) ProductionSNPCallerPlugin

./tassel-5-standalone/run_pipeline.pl -fork1 -ProductionSNPCallerPluginV2 -db Tassel5GBSv2Pipeline/GooseGBS.db -e EcoT22I -mnQS 20 -i ./Tassel5GBSv2Pipeline -k ./Tassel5GBSv2Pipeline/Goose_Keyfile_Tassel5GBSv2Pipeline.txt -kmerLength 64 -o ./Tassel5GBSv2Pipeline/GooseGBS.vcf -endPlugin -runfork1



Filtering with vcftools

#remove indels
vcftools --vcf GooseGBS.vcf --remove-indels --recode --recode-INFO-all --out SNPs_only


#keep only biallelic SNPs
vcftools --vcf SNPs_only.recode.vcf --min-alleles 2 --max-alleles 2 --recode --recode-INFO-all --out BiAllelic_SNPs_only 


#keep SNPs with MAF 0.05 or higher  
vcftools --vcf BiAllelic_SNPs_only.recode.vcf --maf 0.05 --recode --recode-INFO-all --out Filtered_Goose_SNPs