broadinstitute / getzlab-pcawg-mutsig2cv_nc

MutSig2CV for noncoding analysis as run for the PCAWG drivers paper (Rheinbay et al. 2020)

MutSig2CV_NC (PCAWG)

MutSig2CV (Lawrence et al., 2014) adapted for noncoding significance analysis, as run for the PCAWG drivers paper (Rheinbay et al., 2020).

Installing

MutSig is implemented in MATLAB. If you have a MATLAB installation and wish to run MutSig interactively on the MATLAB console, skip to the Running section below. If you do not have MATLAB installed, or do not wish to run interactively, MutSig can be run as a standalone executable. The standalone executable is available for 64 bit Linux systems only, and requires that the MATLAB R2016a runtime be installed. You can download and install the runtime environment from here. Runtime installation instructions can be found here.

Once the runtime is successfully installed, you must add it to your LD_LIBRARY_PATH.

MCRROOT=<path to runtime you specified when installing>
export LD_LIBRARY_PATH=.:${MCRROOT}/runtime/glnxa64
export LD_LIBRARY_PATH=${LD_LIBRARY_PATH}:${MCRROOT}/bin/glnxa64
export LD_LIBRARY_PATH=${LD_LIBRARY_PATH}:${MCRROOT}/sys/os/glnxa64
export LD_LIBRARY_PATH=${LD_LIBRARY_PATH}:${MCRROOT}/sys/opengl/lib/glnxa64

To run on the cohort in the manuscript, MutSig requires ~100 GB of reference files. This is due to the fact that each cohort has a unique set of covariates and coverage models (see "Cohort Parameters" below). Since these files are too large to include in a GitHub repository, they are hosted in cloud storage; please see the README in the ref/ folder for instructions on how to obtain them.

Running

To run interactively on the MATLAB console, start MATLAB in this directory, and run:

MutSig2CV_NC(<path to mutations>, <path to output directory>, <path to cohort parameters>)

To run the standalone application, cd to this directory, and run:

bin/MutSig2CV_NC <path to mutations> <path to output directory> <path to cohort parameters>

MutSig looks for its reference files relative to this directory, so it is essential it is run here. As stated previously, the standalone application will only work on 64 bit Linux systems. As distributed, the interactive version will also only work on 64 bit Linux, but can be made to work on any platform as long as all supplied .mex C/C++ extensions are recompiled.

Each input is decribed below.

Description of inputs

• Mutations file: Absolute path to the set of mutations to analyze. Format is specified in the following section, Mutation Input Format

• Output directory: Absolute path to the directory where MutSig will save its output. Will be created if necessary. NB: Any previous MutSig results in this directory will be overwritten!

• Cohort parameters: Each cohort and noncoding feature (e.g., promoters, UTRs, enhancers, etc.) analyzed in the manuscript has its own set of reference files. This is due to the fact that genomic coverage in noncoding regions can vary substantially from cohort to cohort, and MutSig requires an accurate estimate of sequencing coverage to properly compute mutation densities. MutSig must be explicitly told which cohort/feature-specific reference files to use, which are located in run/params. For example, to analyze transcription factor binding sites in the glioblastoma cohort, use run/params/CNS-GBM_TFBS.params.txt. Cohort parameter files' names follow the convention <cohort>_<feature>.txt, where <cohort> corresponds to the "Tier 3 Abbreviations" in the PCAWG tumor subtype table, and <feature> corresponds to the following noncoding features:

  • enhancers: Enhancers
  • gc_pc.3utr: 3' UTRs
  • gc_pc.5utr: 5' UTRs
  • lncrna: lncRNA genes
  • lncrna.prom: lncRNA promoters
  • mirna.mat: Mature miRNAs
  • mirna.pre: Pre-miRNAs
  • mirna.prom: miRNA promoters
  • promoters: Coding gene promoters
  • TFBS: Transcription Factor Binding Sites

Mutation Input Format

As input, MutSig takes a tab-delimited file with each line annotating a single mutation in a single patient. Columns can be in any order, with names and formats as follows. To provide maximal input flexibility, MutSig accepts synonyms for each column name. Column names are case sensitive.

• chr: Chromosome of the mutation. MutSig only analyzes mutations on autosomal or sex chromosomes, and does not consider the mitochondrial chromosome or unplaced/alternate contigs.

  • Range: (chr)?[1..24XY]
  • Synonyms: Chromosome

• pos: hg19 position of the mutation, 1-indexed.

  • Regex: [0-9]+
  • Synonyms: Position, start, Start_position

• patient: Unique identifier for the patient.

  • Regex: [A-Za-z0-9]+
  • Synonyms: Tumor_Sample_Barcode, Patient_name

• ref_allele: hg19 reference base(s) for the position. In the case of insertions, must be "-".

  • Regex: (-|[ACGT]+)
  • Synonyms: Reference_Allele

• newbase: Observed variant allele at the position. In the case of deletions, must be "-".

  • Regex: (-|[ACGT]+)
  • Synonyms: Tumor_Allele, Tum_allele, Alt_allele, Alternate_allele, Tumor_Seq_Allele2

Note that MutSig does not require any other mutation annotations; it infers everything else on its own.