#requries bcf tools, samtools, and bowtie2 (or other read mapper) bowtie2-build ref.fasta refname #makes bowtie index for reference genome bowtie2 -x refname -1 read1.fq -2 read2.fq -U unpairedread.fq -S output.sam #map reads back to reference genome for sam file samtools view -bS output.sam > output.bam #convert sam to bam samtools sort output.bam -O output.sort.bam #sort by genomic location #need to do next step or the variant annotations are useless samtools mpileup --min-BQ number 20-30 \ -f ref.fasta --BCF bamfromprevious.bam | \ bcftools call --consensus-caller \ --variants-only --pval-threshold 0.05 recommended -Ov -Ob > outputsvariants.bcf ; #gets the variants from read mapping file bcftools norm -m-any outputsvariants.bcf | bcftools norm -Ov --check-ref w -f ref.fasta > outputsvariants.vcf ; #convert to vcf based off of alignment with refernce genome bcftools view outputsvariants.vcf | vcfutils.pl varFilter -d 18 -w 1 -W 3 -a 1 \ -e 0.05 -p > outputsvariants.filtered.vcf ; #filters those variants that don't meet a certain threshold