GBS

Pipeline for analyzing Genotyping-By-Sequencing pair-end data (NGS technique based on digest by restiction enzyme).

M. Fracassetti et al. (2015). Validation of pooled whole-genome re-sequencing in Arabidopsis lyrata.

• Demultiplexing with preprocess_radtags (Stacks), based on barcode and cutting site of the restriction enzyme.
• Trimming with trim-fastq.pl (PoPoolation).
• Alignment to Arabidopsis lyrata genome v1.0 (BWA-MEM).
• Removal of duplicates (Picard tools).
• Selection of reads with MAPQ >20 (SAMtools).
• SNP calling with VarScan.

Software used:

• process_radtags (Stacks, Catchen et al. 2013)
• trim-fastq.pl (PoPoolation, Kofler et al. 2012)
• bwa mem (BWA Li et al. 2013)
• samtools (SAMtools, Li et al. 2009)
• SortSam.jar (Picard tools, http://picard.sourceforge.net)
• MarkDuplicates.jar (Picard tools, http://picard.sourceforge.net)
• genomeCoverageBed (BEDTools, Quinlan & Hall 2010)
• mpileup2snp (VarScan, Kobolt et al. 2012)

Run the following scripts:

1. GBS_demultiplex.sh dempultiplexing and trimming of the fastq files.

2. GBS_alignment.sh Alignment, removal of duplicates, selection of properly aligned reads. Coverage calculations. Creation of bam files for each sample.

3. GBS_SNPcall.sh SNP calling with VarScan.