SNP Calling for GBS Data Pipeline

This pipeline is a prototype of calling SNPs using Genotyping-by-sequencing (GBS) data

Software prerequisites

1) Stacks 2) BWA 3) samtools 4) Picard 5) VCFTools 6) Trimmomatic

Data/resources

1) GBS sequencing data (fastq file) 2) Reference genome (fasta file) 3) Barcodes ID for GBS sequence (txt file) 4) Population map for all dataset (txt file)

Installation of stacks

cd ~/opt
wget https://catchenlab.life.illinois.edu/stacks/source/stacks-2.62.tar.gz
tar xfvz stacks-2.xx.tar.gz
cd stacks-2.xx
./configure --prefix=/path/to/opt/stacks-2.xx
make -j 10

Prepare for the dataset

trim and demultiplex the fastq file for the SNP calling

Trim based on quality and adaptors

trimmomatic-0.39.jar -d PE -fq FileNameSeed -t 10 -ph 33 -ad TruSeq3-PE.fa:2:30:10 -l 30 -sl 4:30 -tr 30 -m 32

Demultiplex (scripts modified from GBS-SNP-CROP)

# Demultiplexing Paired-End (PE) reads:
perl GBS-Demultiplex.pl -d PE -b BarcodeID.txt -fq FileNameSeed
# Demultiplexing Single-End (SE) reads:
perl GBS-SNP-CROP-3.pl -d SE -b BarcodeID.txt -fq FileNameSeed

Align reads to reference genome

bwa index Pcr.genome.1.0.fasta
bash GBS-bwa.sh

SNP calling using stacks

Optinal using coordinate sorted bam file with readgroups added to the BAM header.

Sort the bam files and add readgroups (required samtools and picard)

add readgroupsID readgroups readgroups-lane readgroups-sequence-method information to the bam files

bash runSort-and-addReadgroups.sh

Run gstacks for all sorted bam files

~/opt/stacks-2.62/gstacks -I ./ -M /path/to/pop-map/list -t 20 -O /path/to/output/b-gstacks/

Run populations

analyze a population of individual samples computing a number of population genetics statistics.

~/opt/stacks-2.62/populations -t 20 -M /path/to/pop-map/list -P /path/to/gstacks/b-gstacks/ -O /path/to/output/c-populations --vcf --phylip --batch-size 10

The results are in the c-population directory:

populations.fixed.phylip populations.fixed.phylip.log populations.haplotypes.tsv populations.hapstats.tsv populations.haps.vcf populations.log populations.log.distribs populations.markers.tsv populations.snps.vcf populations.sumstats.tsv populations.sumstats_summary.tsv