Stacks_denovo

Using Stacks (de novo) pipelineto call SNPs with GBS raw data of 94 ponderosa pine (Pinus ponderosa)

Software:

• Version: Stacks Version 2.2
• Website: http://catchenlab.life.illinois.edu/stacks/
• Manual: http://catchenlab.life.illinois.edu/stacks/manual/

Input File

• Raw fasta file produced by GBS (Genotyping by sequencing) with restriction enzyme ApeKI
• Barcode information

Output File

VCF file

Step1: clean the data

• Code: S1_radtags_05.sh, S1_radtags_06.sh
• Input: two raw fasta filet, barcode file
• sliding window: 0.2
• score limit: 20
• maximum read length: 64
• Output: 96 .fq.gz file

Step2: ustack

• Code: S2_ustacks_05.sh, S2_ustacks_06.sh
• Input: 96 .fq.gz file
• Min depth of coverage to create a stack (m): 3
• Max distance allowed between stacks (M): 2
• Max distance allowed to align secondary reads: 4
• Max number of stacks allowed per de novo locus: 3
• Minimum alignment length: 0.8
• Alpha significance level for model: 0.05
• Output: 96 .alleles.tsv.gz file, 96 .tags.tsv.gz file, 96 .snp.tsv.gz file

Step3: cstack

• Code: S3_cstacks.sh
• Input: output file of step 2
• Output: one catalog .alleles.tsv.gz file, one catalog .tags.tsv.gz file, one catalog .snp.tsv.gz file

Step4: sstack

• Code: S4_sstacks.sh
• Input: output files of step 2 and 3
• Output: 96 .matches.tsv.gz file

Step5: tsv2bam

• Code: S5_tsv2bam.sh
• Input: output files of step 2, 3 and 4
• Output: 96 .matches.bam file

Step5.1: sort bam files

• Code: S5.1_sort_bam.sh
• Input: 96 .matches.bam file
• Output: 96 sorted .matches.bam file

Step6: gstacks_denovo

• Code: S6_gstacks_denovo.sh
• Input: 96 sorted .matches.bam file
• Output: one .log.distribs file, one catalog.fa.gz,one catalog.calls

Step7: populations_denovo

• Code: S7_populations_denovo.sh
• Input: output file of Step 6
• Output: VCF file