Abstract

This work shows the process of creating a bam file and vcf from fastq. Three tools are used: bwa, samtools, and gatk. The final file created is VCF. The gVCF contains records for all positions with and without mutation detection.

##Procedure ・Align fastq with bwa to create a sam file.

bwa mem -R "@RG\tID:L\tSM:"${sample1}"\tPL:illumina\tLB:lib1\tPU:unit1" -t 16\ -M ${ref_fasta} ${read1_fastq} ${read2_fastq}

${sample1}.sam

${sample1} : sample name ${ref_fasta} : Specify the location where the reference fasta　(ex ./input/Homo_sapiens_assembly38.fasta) ${read1_fastq},{read2_fastq} : Specify the location where the read fastq (ex ./input/read1_fastq)

・Sort the sam file using samtools and create a bam file.

samtools sort -@4 >${sample1}.sam>${sample1}.sort.bam

samtools index ${sample1}.sort.bam

※Specify the location where files are located.

・Remove duplicates from the bam file using GATK MarkDuplication.

gatk MarkDuplicates -I ${sample1}.sort.bam -M metrics.txt -O ${sample1}.MarkDup.bam --CREATE_INDEX

-I : input file

-O : output

・Mutation detection using GATK HaplotypeCaller

gatk HaplotypeCaller -R ${ref_fasta} -I ${sample1}MarkDup.bam -O {sample1}.output.g.vcf.gz -ERC GVCF

Specify the name of the input and output file

・gVCF to vcf

gatk GenotypeGVCFs --variant ${sample1}.haplotypecaller1.g.vcf -R　${ref_fasta} -O ${sample1}.haplotypecaller.vcf

Specify the name of the input and output file

※For multiple samples

gatk GenotypeGVCFs
　　--variant haplotypecaller1.g.vcf
　　--variant haplotypecaller2.g.vcf
　　--variant haplotypecaller3.g.vcf
　　-R ${ref_fasta}
　　-O ${sample1}.haplotypecaller.vcf