Mutect2 Somatic Variant Calling Module

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Overview

This is a GenePattern module wrapping GATK Mutect2. This module supports optional use of a germline reference, panel of normals (PoN), intervals, and parallel scatter–gather execution.

Call somatic short mutations via local assembly of haplotypes. Short mutations include single nucleotide (SNA) and insertion and deletion (indel) alterations. The caller uses a Bayesian somatic genotyping model that differs from the original MuTect by Cibulskis et al., 2013 and uses the assembly-based machinery of HaplotypeCaller.

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Features

• Tumor vs. matched Normal somatic variant calling
• Support for Germline Resource (e.g. gnomAD)
• Support for Panel of Normals (PoN)
• Optional interval-based calling (BED or interval list)
• Optional parallel processing (scatter–gather)
• Produces both unfiltered and filtered VCFs
• Outputs Mutect2 filtering and contamination metrics

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Input Files

1. Tumor BAM (required)

Coordinate-sorted BAM of the tumor sample with .bai index.

2. Reference Genome FASTA (required)

Must include .fai and .dict index files.

3. Normal BAM (optional but recommended)

Matched normal BAM with index.

4. Germline Resource (VCF) (optional but recommended)

Population allele-frequency resource such as gnomAD.

5. Panel of Normals (PoN) (optional but recommended)

A PoN VCF identifying recurrent technical artefacts.

6. Intervals (BED or .interval_list) (optional)

Used to restrict calling to target regions.

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Output Files

File	Description
<OutputName>.unfiltered.vcf.gz	Raw candidate variants
<OutputName>.vcf.gz	Filtered somatic variants
<OutputName>.vcf.gz.tbi	Tabix index
<OutputName>_metrics.txt	Filter stats & QC metrics
(Optional) intermediate scatter VCFs	Only if parallel mode enabled

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Parameters

Parameter	Description	Required
Reference	Reference genome FASTA	Yes
Tumor BAM	BAM file for tumor sample	Yes
Tumor Sample	SM tag for tumor	Yes
Output Name	Prefix for output files	Yes
Normal BAM	Matched normal BAM	No
Normal Sample	SM tag for normal	Required if Normal BAM provided
Germline Resource	gnomAD or similar VCF	No
Panel of Normals	PoN VCF	No
Intervals	Regions to call variants	No
Parallel Processing	"Yes" or "No"	Yes

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Parallel Processing (Scatter–Gather)

If enabled, the module:

1. Splits genomic intervals into chunks
2. Runs parallel Mutect2 calls
3. Gathers VCFs into a single unified result

Recommended for WGS or large targeted panels. Can cause boundary defects in output

If disabled, Mutect2 runs a single job across all regions.

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Sample Data

Tumor & Normal Reads: FastQ/BAM data for tumor and normal samples from GIAB (HG008, Liss_lab) via FTP:

https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data_somatic/HG008/Liss_lab/NYGC_Illumina-WGS_20231023/

Reference Genome: hg38 assembly:

"https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz"

Germline Resource

https://storage.googleapis.com/gcp-public-data--broad-references/hg38/v0/somatic-hg38/af-only-gnomad.hg38.vcf.gz

Pannel of Normals

https://storage.googleapis.com/gatk-best-practices/somatic-hg38/1000g_pon.hg38.vcf.gz

Intervals List

https://storage.googleapis.com/gcp-public-data--broad-references/hg38/v0/exome_calling_regions.v1.1.interval_list

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References

GATK Mutect2 documentation https://gatk.broadinstitute.org/hc/en-us/articles/360037593851-Mutect2

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Version

Mutect2 Module — v1.0