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Workflow summary

Tools for Alignment workflowTools for Alignment workflow

The BWA-MEM algorithm is used in the map/alignment step, and directly converted into a bam file using Samtools. Tumor and normal samples are labeled separately in read groups, and sorted by chromosome using GATK-Picard’s Sortsam. Duplication marks and base recalibration are then performed using GATK. See below for detailed command-line scripts.

DNA Sequencing Analysis GuideReference Genome

All sequence reads were aligned using the human reference genome hg38 which is the UCSC modified version of GRCh38.p14 released in June 2022. More information is here.

DNA sequencing pipeline command-line parameters

Step 1 : Map/Align (BWA-MEM)

bwa mem -t <Num threads> \ -R <read group> \ <reference fasta> \ <R1 fastq> <R2 fastq> | \ samtools view -Shb –o <out bam>

Step 2 : Sort bam (GATK-Picard)

gatk SortSam \ -I <in bam> \ -O <out bam> \ -SO coordinate \ --VALIDATION_STRINGENCY SILENT \ --MAX_RECORDS_IN_RAM 50000000 && \ samtools index <out bam>

Step 3 : Mark duplicate (GATK)

gatk MarkDuplicates \ -I <in bam> \ -O <marked_dup bam> \ -M <marked_dup metrics> \ --VALIDATION_STRINGENCY SILENT \ --MAX_RECORDS_IN_RAM 50000000 && \ samtools index <marked_dup bam>

Step 4 : Recalibrate Base Quality Scores (GATK)

gatk BaseRecalibrator \ -R <reference fasta> \ -I <in bam> \ --known-sites <known sites vcf> \ -O <recal table>
gatk ApplyBQSR \ -R <reference fasta> \ -I <in bam> \ --bqsr-recal-file <recal table> \ -O <recalibrated bam> \ --create-output-bam-index false && \ samtools index <recalibrated bam>

Tools for Somatic Mutation call workflow

The map/aligned bam files are analyzed for somatic mutations using four tools: Mutect2, MuSE, VarScan2, and Pindel. Mutation calls are reported by each tool in a VCF file.

Somatic Mutation call pipeline command-line parameters

Mutect2 (GATK) Germline resource: af-only-gnomad.vcf.gz Panel of normals: pon.vcf.gz

gatk Mutect2 \ -R <reference fasta> \ -I <tumor bam> \ -I <normal bam> \ -normal normal \ --germline-resource <af-only-gnomad.vcf.gz> \ --panel-of-normals <pon.vcf.gz> \ -O <out vcf>

MuSE dbSNP: dbsnp155

# MuSE call Step MuSE call \ <Tumor bam> <Normal bam>\ -f <reference fasta>\ -O <muse call intermediate>
# MuSE sump Step MuSE sump \ -I <muse call intermediate>\ -G \ # -G and -E stand for WGS and WES, respectively -D <dbsnp155.vcf.gz>\ -O <out vcf>

VarScan2

# Mpileup Step via samtools -f <reference fasta> \ -q 0 \ -B \ <Normal bam> <Tumor bam> \ <intermediate_mpileup.pileup>
# Somatic mutation call varscan somatic \ <intermediate_mpileup.pileup> \ <out prefix> \ --mpileup 0 \ --min-coverage 8 \ --min-coverage-normal 8 \ --min-coverage-tumor 6 \ --min-var-freq 0.10 \ --min-freq-for-hom 0.75 \ --normal-purity 1.0 \ --tumor-purity 1.00 \ --p-value 0.99 \ --somatic-p-value 0.05 \ --strand-filter 0 \ --output-vcf 1
# Mutation classification varscan processSomatic \ <intermediate vcf> \ --min-tumor-freq 0.10 \ --max-normal-freq 0.05 \ --p-value 0.07

Pindel

sambamba view <Tumor bam> \ --filter "\not (unmapped or duplicate or secondary_alignment or failed_quality_control or supplementary)\" \ --format bam \ --output-filename <Tumor sambamba bam> \ gatk CollectInsertSizeMetrics \ -I <Tumor sambamba bam> \ -O <Tumor_insert_size_metric.txt> \ -H <Tumor_insert_size_metric.pdf> && \ sambamba view <Normal bam> \ --filter "\not (unmapped or duplicate or secondary_alignment or failed_quality_control or supplementary)\" \ --format bam \ --output-filename <Normal sambamba bam> \ gatk CollectInsertSizeMetrics \ -I <Normal sambamba bam> \ -O <Normal_insert_size_metric.txt> \ -H <Normal_insert_size_metric.pdf> && \ t_size=$(printf "%.0f" "$(head -8 <Tumor_insert_size_metric.txt> | tail -1 | awk 'BEGIN{FS=OFS="\t";}{print $6;}')") \ n_size=$(printf "%.0f" "$(head -8 <Normal_insert_size_metric.txt> | tail -1 | awk 'BEGIN{FS=OFS="\t";}{print $6;}')") \ echo -e "<Tumor sambamba bam>\t\ttumor" > <Tumor>_<Normal>.pindel.config \ echo -e "<Normal sambamba bam>\t\tnormal" >> <Tumor>_<Normal>.pindel.config && \ pindel \ -f <reference fasta> \ -i <Tumor>_<Normal>.pindel.config \ -o <Output prefix> && \ grep ChrID <Output prefix>.D > <Output prefix>.D.head \ grep ChrID <Output prefix>.SI > <Output prefix>.SI.head && \ cat <Output prefix>.D.head <Output prefix>.SI.head > <Output prefix>.pindel_somatic && \ cat << EOF > <Output prefix>.somatic.indel.filter.config indel.filter.input = <Output prefix>.pindel_somatic indel.filter.vaf = 0.08 indel.filter.cov = 20 indel.filter.hom = 6 indel.filter.pindelvcf = <PINDEL path> indel.filter.reference = <reference fasta> indel.filter.referenceindex = <genome build> indel.filter.referencedate = $(date '+%Y%m%d') indel.filter.output = <Output prefix>_filter.vcf EOF && \ perl <PINDEL path>/bin/somatic_filter/somatic_indelfilter.pl <Output prefix>.somatic.indel.filter.config && \ gatk SortVcf \ -I <Output prefix>_filter.vcf \ -O <Output prefix>_filter_sorted.vcf \ -SD <reference dictionary file> \ --CREATE_INDEX false && \ tabix -p vcf <Output prefix>_filter_sorted.vcf.gz && \ gatk VariantFiltration \ -R <Reference fasta> \ -V <Output prefix>_filter_sorted.vcf.gz \ -O <Output prefix>_filtered.vcf.gz \ --filter-name TALIDP \ --filter-expression "\(vc.isbiallelic() && vc.getGenotype(\"TUMOR\").getAD().1 < 3\)" \ --create-output-variant-index false && \ tabix -p vcf <Output prefix>_filtered.vcf.gz

Mutation annotation command line parameters vep cache : Ensembl v.111

vcf2maf.pl --input-vcf <Tumor-Normal vcf> \ --output-maf /root/output/$2_varscan.hc.maf \ --tumor-id <tumor sample ID> \ --normal-id <Normal sample ID> \ --vcf-tumor-id TUMOR \ --vcf-normal-id NORMAL \ --ref-fasta <reference fasta> \ --cache-version 111 \ --ncbi-build GRCh38

RNA Sequencing Analysis Guide

Methylation Sequencing Analysis Guide

Methylation Array Analysis Guide