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mutect-nf

Mutect pipeline for somatic variant calling with Nextflow

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Description

Nextflow pipeline for somatic variant calling with mutect with Mutect1 or 2, gatk3 or gatk4

Dependencies

  1. Nextflow: for common installation procedures see the IARC-nf repository.
  2. Mutect and its dependencies (Java 1.7 and Maven 3.0+), or gatk4 that now includes Mutect2
  3. bedtools and move the executable file in your path.
  4. python and package pysam
  5. bedops

A conda receipe, and docker and singularity containers are available with all the tools needed to run the pipeline (see "Usage")

GATK4

With GATK4, a list of known_snps can be provided to mutect2 to improve the variant classification, for example file af-only-gnomad.hg38.vcf.gz from the bundle best practices from the broad institute GATK somatic calling bundle.

estimate contamination

When the estimate contamination mode is chosen, one needs to provide a list of known snps; we recommend the file small_exac_common_3.hg38.vcf.gz from the best practices broad institute bundle.

Input

Type Description
--tumor_bam_folder a folder with tumor bam files
--normal_bam_folder a folder with normal bam files
--tn_file input tabulation-separated values file with columns sample (sample name), tumor (full path to tumor bam), normal (full path to matched normal bam); optionally (for --genotype mode), columns preproc (is the bam RNAseq needing preprocessing: yes or no) and vcf (full path to vcf file containing alleles to genotype)

Input methods

Note that there are two input methods: separate tumor_bam_folder and normal_bam_folder, and tn_file.

Separated tumor_bam_folder and normal_bam_folder method

The method assumes that normal and tumor bam files are in these respective folder, and uses parameters suffix_tumor and suffix_normal to detect them (the rest of the file name needs to be identical.

The tumor bam file format must be (sample suffix_tumor .bam) with suffix_tumor as _T by default and customizable in input (--suffix_tumor). (e.g. sample1_T.bam) The normal bam file format must be (sample suffix_normal .bam) with suffix_normal as _N by default and customizable in input (--suffix_normal). (e.g. sample1_N.bam). BAI indexes have to be present in the same location than their BAM mates, with the extension bam.bai.

The tn_file method

The method uses a tabulation-separated values format file with columns sample, tumor, and normal (in any order); it does not use parameters suffix_tumor and suffix_normal and does not require file names to match. When the genotype mode is active, additional columns are expected: preproc, specifying if preprocessing of RNA-seq bam file is required (yes or no) and vcf, indicating the location of the vcf file containing the alleles to genotype. preproc includes splitting spanning reads, correcting CIGAR string with NDN pattern, and changing mapping quality of uniquely mapped reads from 255 to 60(gatk4's splitNCigarReads and a custom python script). The tn_file method is necessary for joint multi-sample calling, in which case the sample name is used to group files, and to specify preprocessing of some RNA-seq samples.

BAI indexes have to be present in the same location than their BAM mates, with the extension bam.bai.

Parameters

  • Mandatory

Name Example value Description
--ref ref.fa reference genome fasta file
  • Optional

Name Default value Description
--cpu 4 number of CPUs
--mem 8 memory for mapping
--suffix_tumor _T suffix for tumor file
--suffix_normal _N suffix for matched normal file
--output_folder mutect_results output folder for aligned BAMs
--bed Bed file containing intervals
--region A region defining the calling, in the format CHR:START-END
--known_snp VCF file with known variants and frequency (e.g., from gnomad)
--mutect_args Arguments you want to pass to mutect. WARNING: form is " --force_alleles " with spaces between quotes
--nsplit 1 Split the region for calling in nsplit pieces and run in parallel
--java java Name of the JAVA command
--snp_contam VCF file with known germline variants to genotype for contamination estimation (requires --estimate_contamination)
--PON path to panel of normal VCF file used to filter calls
--gatk_version 4 gatk version
--ref_RNA fasta reference for preprocessing RNA (required when preproc column contains yes in input tn_file)

NOTE: if neither --bed or --region, will perform the calling on whole genome, based on the faidx file.

  • Optional for gatk3

These options are not needed if gatk4 is used

Name Default value Description
--cosmic Cosmic VCF file required by mutect; not in gatk4
--mutect_jar path to jar file of mutect1
--mutect2_jar path to jar file of mutect2
  • Flags

Name Description
--help print usage and optional parameters
--estimate_contamination run extra step of estimating contamination by normal and using the results to filter calls; only for gatk4
--genotype use genotyping from vcf mode instead of usual variant calling requires tn_file with vcf column and gatk4, and if RNA-seq included, requires preproc column
--filter_readorientation Run extra step learning read orientation model and using it to filter reads

Usage

To run the pipeline on a series of matched tumor normal files (with suffixes _T and _N) in folders tumor_BAM normal_BAM, a reference genome with indexes ref, and a bed file ref.bed, one can type:

nextflow run IARCbioinfo/mutect-nf -r v2.2b -profile singularity  --tumor_bam_folder tumor_BAM/ --normal_bam_folder normal_BAM/ --ref ref_genome.fa --gtf ref.gtf 

To run the pipeline without singularity just remove "-profile singularity". Alternatively, one can run the pipeline using a docker container (-profile docker) the conda receipe containing all required dependencies (-profile conda). Note that we provide similar support when using gatk3 (profiles conda_gatk3, singularity_gatk3, and docker_gatk3) or gatk2 (profiles conda_gatk2, singularity_gatk2, and docker_gatk2).

To use gatk3, set --gatk_version 3and provide option --mutect2_jar for mutect version 2 (GATK executable jar, which integrate mutect2) and possibly specify -profile singularity_gatk3, and set --mutect_jar for mutect version 1 and possibly specify -profile singularity_gatk2.

Help section

You can print the help manual by providing --help in the execution command line:

nextflow run iarcbioinfo/mutect-nf --help

This shows details about optional and mandatory parameters provided by the user.

Output

Type Description
sample.vcf.gz and sample.vcf.gz.tbi filtered VCF files and their indexes
stats/ gatk stats files from mutect
intermediate_calls/raw_calls/sample.vcf unfiltered VCF files

The output_folder directory contains two subfolders: stats and intermediate_calls

FAQ

Why are some samples absent from the output vcfs when I run multi-sample calling?

Outputs are based on the SM field of the BAM file; when multiple files have the same SM, only one is outputed.

Why are some samples present in the input file ignored?

Check that the input is tab-separated. When parsing the input file, if a line is not tab separated, nextflow will ignore it without returning an error.

Directed Acyclic Graph

DAG

Contributions

Name Email Description
Nicolas Alcala* [email protected] Developer to contact for support
Tiffany Delhomme Developer