Table of contents

signeR

Rodrigo Drummond, Renan Valieris, Rafael Rosales and Israel Tojal da Silva

1: Introduction

Motivation: Cancer is an evolutionary process driven by continuous acquisition of genetic variations in individual cells. The diversity and complexity of somatic mutational processes is a conspicuous feature orchestrated by DNA damage agents and repair processes, including exogenous or endogenous mutagen exposures, defects in DNA mismatch repair and enzymatic modification of DNA. The identification of the underlying mutational processes is central to understanding of cancer origin and evolution.

The signeR package focuses on the estimation and further analysis of mutational signatures. The functionalities of this package can be divided into three categories. First, it provides tools to process VCF files and generate matrices of SNV mutation counts and mutational opportunities, both defined according to a 3bp context (mutation site and its neighbouring 3' and 5' bases). Second, these count matrices are considered as input for the estimation of the underlying mutational signatures and the number of active mutational processes. Third, the package provides tools to correlate the activities of those signatures with other relevant information such as clinical data, in order to draw conclusions about the analysed genome samples, which can be useful for clinical applications. These include the Differential Exposure Score and the a posteriori sample classification.

Although signeR is intended for the estimation of mutational signatures, it actually provides a full Bayesian treatment to the non-negative matrix factorisation (NMF) model. Further details about the method can be found in Rosales & Drummond et al., 2016 (see section 6.1 below).

This vignette briefly explains the use of signeR through examples.

2: Installation

Before installing, please make sure you have the latest version of R and Bioconductor installed.

To install signeR, start R and enter:

install.packages("BiocManager")
BiocManager::install("signeR")

For more information, see this page.

Once installed the library can be loaded as

library(signeR)

OS X users might experience compilation errors due to missing gfortran libraries. Please read this section.

3: Preparing the input

signeR takes as input a count matrix of samples x features. Each feature is usually a SNV mutation within a 3bp context (96 features, 6 types of SNV mutations and 4 possibilities for the bases at each side of the SNV change). Optionally, an opportunity matrix can also be provided containing the count frequency of the features in the whole analyzed region for each sample. Although not required, this argument is highly recommended because it allows signeR to normalize the features frequency over the analyzed region.

Input matrices can be read both from a VCF or a tab-delimited files, as described next.

3.1: Input from VCF

The VCF file format is the most common format for storing genetic variations, the signeR package includes a utility function for generating a count matrix from the VCF:

library(VariantAnnotation)

# BSgenome, equivalent to the one used on the variant call
library(BSgenome.Hsapiens.UCSC.hg19)

vcfobj <- readVcf("/path/to/a/file.vcf", "hg19")
mut <- genCountMatrixFromVcf(BSgenome.Hsapiens.UCSC.hg19, vcfobj)

This function will generate a matrix of mutation counts for each sample in the provided VCF.

If you have one vcf per sample you can combine the results into a single matrix like this:

mut = matrix(ncol=96,nrow=0)
for(i in vcf_files) {
    vo = readVcf(i, "hg19")
    # sample name (should pick up from the vcf automatically if available)
    # colnames(vo) = i
    m0 = genCountMatrixFromVcf(mygenome, vo)
    mut = rbind(mut, m0)
}
dim(mut) # matrix with all samples

The opportunity matrix can also be generated from the reference genome (hg19 in the following case):

library(rtracklayer)

target_regions <- import(con="/path/to/a/target.bed", format="bed")
opp <- genOpportunityFromGenome(BSgenome.Hsapiens.UCSC.hg19,
    target_regions, nsamples=nrow(mut))

Where target.bed is a bed file containing the genomic regions analyzed by the variant caller.

If a BSgenome is not available for your genome, you can use a fasta file:

library(Rsamtools)

# make sure /path/to/genome.fasta.fai exists !
# you can use "samtools faidx" command to create it
mygenome <- FaFile("/path/to/genome.fasta")

mut <- genCountMatrixFromVcf(mygenome, vcfobj)
opp <- genOpportunityFromGenome(mygenome, target_regions)

3.2: Input from tab-delimited file

By convention, the input file should be tab-delimited with sample names as row names and features as column names. Features should be refered in the format "base change:triplet", e.g. "C>A:TCG", as can be seen in the example below. Similarly, the opportunity matrix can be provided in a tab-delimited file with the same structure as the mutation counts file. An example of the required matrix format can be seen here.

This tutorial uses as input the 21 breast cancer dataset described in Nik-Zainal et al 2012. For the sake of convenience this dataset is included with the package and can be accessed by using the system.file function:

mut <- read.table(system.file("extdata","21_breast_cancers.mutations.txt",
    package="signeR"), header=TRUE, check.names=FALSE)
opp <- read.table(system.file("extdata","21_breast_cancers.opportunity.txt",
    package="signeR"))

3.3: Known signatures

signeR analysis can incorporate any previous knowledge about the signatures present in the dataset. If signatures are known in advance, they can be provided as a matrix, which may be used by signeR in two different ways: a starting value that will be updated according to mutation patterns found on present data or a fixed set of parameters, kept unchanged during the estimation of exposures.

Signatures matrix shall contain each signature in one colum. An example of the required matrix format can be seen here.

Along this tutorial a matrix of signatures found in breast cancer, as described in Cosmic database. For the sake of convenience this matrix is included with the package and can be accessed by the system.file function:

Pmatrix <- as.matrix(read.table(system.file("extdata","Cosmic_signatures_BRC.txt",
    package="signeR"), sep="\t", check.names=FALSE))

4: Estimating the number of mutational processes and their signatures

signeR takes a count matrix as its only required parameter, but the user can provide an opportunity matrix as well. The algorithm allows the assessment of the number of signatures by three options, as follows.

  1. signeR detects the number of signatures at run time by considering the best NMF factorisation rank between 1 and min(G, K)-1, with G = number of genomes and K = number of features (i.e. 96):
    signatures <- signeR(M=mut, Opport=opp)
    
  2. The user can give an interval of the possible numbers of signatures as the parameter nlim. signeR will calculate the optimal number of signatures within this range, for example:
    signatures <- signeR(M=mut, Opport=opp, nlim=c(3,7))
    
  3. signeR can also be run by passing the number of signatures as the parameter nsig. In this setting, the algorithm is faster. For example, the following command will make signeR consider only the rank N=5 to estimate the signatures and their exposures:
    signatures <- signeR(M=mut, Opport=opp, nsig=3, main_eval=100, EM_eval=50, EMit_lim=20)
    
  4. Finally, when signatures are known in advance, signeR can use them as a starting point for the estimation of signatures in the present dataset. To this end, signatures must be provided in a matrix, as described in item 3.3 above. For example, the following command will make signeR use six Cosmic signatures found on breast cancer as a starting point:
    signatures.Pstart <- signeR(M=mut, Opport=opp, P=Pmatrix, fixedP=FALSE, main_eval=100, EM_eval=50, EMit_lim=20)
    

The parameters testing_burn and testing_eval control the number of iterations used to estimate the number of signatures (default value is 1000 for both parameters). There are other arguments that may be passed on to signeR. Please have a look at signeR's manual, issued by typing help(signeR).

Whenever signeR is left to decide which number of signatures is optimal, it will search for the rank Nsig that maximizes the median Bayesian Information Criterion (BIC). After the processing is done, this information can be plotted by the following command:

BICboxplot(signatures)

Boxplot of BIC values, showing that the optimal number of signatures for this dataset is 5.

5: Estimating exposures to known signatures.

signeR also offer the possibility to estimate exposures to known signatures as, for example, the ones described on Cosmic database. In this case, signatures should be provided in a matrix, as described in intem 3.3 above, and should be kept constant during analysis:

Pmatrix <- as.matrix(read.table(system.file("extdata","Cosmic_signatures_BRC.txt",
    package="signeR"), sep="\t", check.names=FALSE))

The following command will make signeR estimate the exposures to the Cosmic signatures found on breast cancer:

exposures.known.sigs <- signeR(M=mut, Opport=opp, P=Pmatrix, fixedP=TRUE, main_eval=100, EM_eval=50, EMit_lim=20)

Exposures can then be recovered from the signer output by the following command (as in any signer analysis):

exposures <- Median_exp(exposures.known.sigs$SignExposures)

6: Results and plots

signeR offer several plots to visualize estimated signatures and their exposures, as weel as the convergence of the MCMC used to estimate them.

6.1: Plot the MCMC paths for the NMF parameters (P and E matrices)

The following instruction plots the MCMC samples paths for each entry of the signature matrix P and their exposures, i.e. the E matrix. Only post-burnin paths are available for plotting. Those plots are useful for checking if entries have leveled off, reflecting the sampler convergence.

Paths(signatures$SignExposures)