Steps for calling variants

1. Trim fastq files - Trimmomatic
2. Reference based mapping - Bowtie2
3. Add or replace read groups in bam file - Picard
   • This tool enables the user to replace all read groups in the INPUT file with a single new read group and assign all reads to this read group in the OUTPUT BAM file
4. Mark duplicates - Picard
   • Identifies duplicate reads
   • This tool locates and tags duplicate reads in a BAM or SAM file, where duplicate reads are defined as originating from a single fragment of DNA
   • Bam file must be indexed for the following GATK command
5. Merge bam files - samtools merge
   • this command is necessary to run Freebayes
   • Freebayes requires one bam file, but it can be merged
6. Run Freebayes to call variants on bam files - Freebayes
7. Filter SNPs and Indels from VCF file output from Freebayes
   • gatk SelectVariants
   • gatk Variant Filtration
8. Recalibrate base quality score - gatk BaseRecalibrator
9. Apply BQSR reports into the bam files - gatk ApplyBQSR
10. Merge recalibrated bam files - samtools merge
11. Run Freebayes to call variants on recalibrated bam files
12. Filter vcf file from freebayes for SNPs only - vcffilter
13. Compressing and indexing the snp only vcf & variant normalization of the snp only vcf & decompressing the vcf
    • compress vcf - bgzip
    • index vcf - tabix
    • normalize snp vcf - bcftools norm
    • decompress the normalized vcf - bgzip
14. Filter normalized vcf file and create SNP alignment fasta file - used custom made scripts
    • vcflib_pipeline_HPC2.sh script
    • uses vcf_fa_extractor.py to filter and make fasta file from vcf