TASSEL_GBS_V2

Using TASSEL GBS V2 pipeline to call SNPs with GBS raw data of 94 ponderosa pine (Pinus ponderosa) and the reference genome of loblolly pine (Pinus taeda).

Software

• Version: TASSEL Version 5.0, GBS V2
• Website: http://www.maizegenetics.net/tassel
• Manual: https://bitbucket.org/tasseladmin/tassel-5-source/wiki/Tassel5GBSv2Pipeline

Input File

• Raw fasta file produced by GBS (Genotyping by sequencing) with restriction enzyme ApeKI
• Reference genome of loblolly pine (Pinus taeda)
• Barcode information

Output File

VCF file

Step 1: fastq to db file

• Code: S1_fqtodb.sh
• Input: Two fasta file & one barcode file
• Plugin: -GBSSeqToTagDBPlugin
• Restriction enzyme: ApeKI
• Minimum Kmer length: 20
• Maximum Kmer length: 64
• Minimum Kmer count: 5
• Minimum count reads: 1
• Output: A .db file

Step 2: db to tag fq file

• Code: S2_dbtotagfq.sh
• Input: .db file
• Plugin: -TagExportToFastqPlugin
• Minimum count reads: 1
• Output: .fa.gz file

Step 3_bwa_1:

• Code: S3_BWA_1.sh
• Input: reference fasta file
• Output: five files

Step 3_bwa_2:

• Code: S3_BWA_2.sh
• Input: .fa.gz file and the five files from Step 3_bwa_1
• Output: .sai file

Step 3_bwa_3:

• Code: S3_BWA_3.sh
• Input: .fa.gz file, the five files from Step 3_bwa_1, and the .sai file
• Output: .sam file

Step 3_bowtie2_1:

• Code: S3_bowtie2_1.sh
• Input: reference fasta file
• Output: five files and .fa.gz file

Step 3_bowtie2_2:

• Code: S3_bowtie2_2.sh
• Input: five files and .fa.gz file from step 3_bowtie2_1
• Output: .sam file

Step 4_samtodb:

• Code: S4_samtodb.sh
• Input: .sam file from step bowtie2_2, .db file from step 2
• Plugin: -SAMToGBSdbPlugin
• Output: updated .db file

Step 5_discSNP:

• Code: S5_discSNP.sh
• Input: updated .db file from Step 4
• Plugin: -DiscoverySNPCallerPluginV2
• -mnLCov : 0.1
• Output: updated .db file

Step 6_prodSNP_vcf:

• Code: S6_prodSNP_vcf.sh
• Input: raw fasta file, barcode file, updated .db file from Step 5
• Kmer length: 64
• Plugin: -ProductionSNPCallerPluginV2
• mnQS: 20
• Output: .vcf file

Step 6_prodSNP_h5:

• Code: S6_prodSNP_h5.sh
• Input:raw fasta file, barcode file, updated .db file from Step 5
• Kmer length: 64
• Plugin: -ProductionSNPCallerPluginV2
• mnQS: 20
• Output: .h5 file