maftools
Summarize, Analyze and Visualize MAF Files

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maftools : Summarize, Analyze and Visualize MAF Files

maftools : Summarize, Analyze and Visualize MAF Files
In maftools: Summarize, Analyze and Visualize MAF Files

Introduction

With advances in Cancer Genomics, Mutation Annotation Format (MAF) is being widely accepted and used to store somatic variants detected. The Cancer Genome Atlas Project has sequenced over 30 different cancers with sample size of each cancer type being over 200. Resulting data consisting of somatic variants are stored in the form of Mutation Annotation Format. This package attempts to summarize, analyze, annotate and visualize MAF files in an efficient manner from either TCGA sources or any in-house studies as long as the data is in MAF format.

Citation

If you find this tool useful, please cite:

Mayakonda A, Lin DC, Assenov Y, Plass C, Koeffler HP. 2018. Maftools: efficient and comprehensive analysis of somatic variants in cancer. Genome Resarch PMID: 30341162

Generating MAF files

MAF field requirements

MAF files contain many fields ranging from chromosome names to cosmic annotations. However most of the analysis in maftools uses following fields.

Complete specification of MAF files can be found on NCI GDC documentation page.

This vignette demonstrates the usage and application of maftools on an example MAF file from TCGA LAML cohort 1.

Installation

if (!require("BiocManager"))
    install.packages("BiocManager")
BiocManager::install("maftools")

Overview of the package

maftools functions can be categorized into mainly Visualization and Analysis modules. Each of these functions and a short description is summarized as shown below. Usage is simple, just read your MAF file with read.maf (along with copy-number data if available) and pass the resulting MAF object to the desired function for plotting or analysis.

Reading and summarizing maf files

Required input files

Reading MAF files.

read.maf function reads MAF files, summarizes it in various ways and stores it as an MAF object. Even though MAF file is alone enough, it is recommended to provide annotations associated with samples in MAF. One can also integrate copy number data if available.

library(maftools)

#path to TCGA LAML MAF file
laml.maf = system.file('extdata', 'tcga_laml.maf.gz', package = 'maftools') 
#clinical information containing survival information and histology. This is optional
laml.clin = system.file('extdata', 'tcga_laml_annot.tsv', package = 'maftools') 

laml = read.maf(maf = laml.maf, clinicalData = laml.clin)

MAF object

Summarized MAF file is stored as an MAF object. MAF object contains main maf file, summarized data and any associated sample annotations.

There are accessor methods to access the useful slots from MAF object.

#Typing laml shows basic summary of MAF file.
laml

````r

Shows sample summry.

getSampleSummary(laml)

Shows gene summary.

getGeneSummary(laml)

shows clinical data associated with samples

getClinicalData(laml)

Shows all fields in MAF

getFields(laml)

Writes maf summary to an output file with basename laml.

write.mafSummary(maf = laml, basename = 'laml')

# Visualization
## Plotting MAF summary.
We can use `plotmafSummary` to plot the summary of the maf file, which displays number of variants in each sample as a stacked barplot and variant types as a boxplot summarized by Variant_Classification. 

```r
plotmafSummary(maf = laml, rmOutlier = TRUE, addStat = 'median', dashboard = TRUE, titvRaw = FALSE)

Oncoplots

Drawing oncoplots

Better representation of maf file can be shown as oncoplots, also known as waterfall plots. 

#oncoplot for top ten mutated genes.
oncoplot(maf = laml, top = 10)

NOTE: Variants annotated as Multi_Hit are those genes which are mutated more than once in the same sample.

For more details on customisation see the Customizing oncoplots vignette.

Transition and Transversions.

titv function classifies SNPs into Transitions and Transversions and returns a list of summarized tables in various ways. Summarized data can also be visualized as a boxplot showing overall distribution of six different conversions and as a stacked barplot showing fraction of conversions in each sample.

laml.titv = titv(maf = laml, plot = FALSE, useSyn = TRUE)
#plot titv summary
plotTiTv(res = laml.titv)

Lollipop plots for amino acid changes

lollipopPlot function requires us to have amino acid changes information in the maf file. However MAF files have no clear guidelines on naming the field for amino acid changes, with different studies having different field (or column) names for amino acid changes. By default, lollipopPlot looks for column AAChange, and if its not found in the MAF file, it prints all available fields with a warning message. For below example, MAF file contains amino acid changes under a field/column name 'Protein_Change'. We will manually specify this using argument AACol. 

#lollipop plot for DNMT3A, which is one of the most frequent mutated gene in Leukemia.
lollipopPlot(maf = laml, gene = 'DNMT3A', AACol = 'Protein_Change', showMutationRate = TRUE)

By default lollipopPlot uses the longest isoform of the gene.

Labelling points.

We can also label points on the lollipopPlot using argument labelPos. If labelPos is set to 'all', all of the points are highlighted. 

lollipopPlot(maf = laml, gene = 'DNMT3A', showDomainLabel = FALSE, labelPos = 882)

Rainfall plots

Cancer genomes, especially solid tumors are characterized by genomic loci with localized hyper-mutations 5. Such hyper mutated genomic regions can be visualized by plotting inter variant distance on a linear genomic scale. These plots generally called rainfall plots and we can draw such plots using rainfallPlot. If detectChangePoints is set to TRUE, rainfall plot also highlights regions where potential changes in inter-event distances are located. 

brca <- system.file("extdata", "brca.maf.gz", package = "maftools")
brca = read.maf(maf = brca, verbose = FALSE)

rainfallPlot(maf = brca, detectChangePoints = TRUE, pointSize = 0.4)

"Kataegis" are defined as those genomic segments containing six or more consecutive mutations with an average inter-mutation distance of less than or equal to 1,00 bp 5.

Compare mutation load against TCGA cohorts

tcgaCompare uses mutation load from TCGA MC3 for comparing muttaion burden against 33 TCGA cohorts. Plot generated is similar to the one described in Alexandrov et al 5. 

laml.mutload = tcgaCompare(maf = laml, cohortName = 'Example-LAML', logscale = TRUE, capture_size = 50)

Plotting VAF

This function plots Variant Allele Frequencies as a boxplot which quickly helps to estimate clonal status of top mutated genes (clonal genes usually have mean allele frequency around ~50% assuming pure sample)

plotVaf(maf = laml, vafCol = 'i_TumorVAF_WU')

Processing copy-number data

Reading and summarizing gistic output files.

We can summarize output files generated by GISTIC programme. As mentioned earlier, we need four files that are generated by GISTIC, i.e, all_lesions.conf_XX.txt, amp_genes.conf_XX.txt, del_genes.conf_XX.txt and scores.gistic, where XX is the confidence level. See GISTIC documentation for details.

all.lesions <- system.file("extdata", "all_lesions.conf_99.txt", package = "maftools")
amp.genes <- system.file("extdata", "amp_genes.conf_99.txt", package = "maftools")
del.genes <- system.file("extdata", "del_genes.conf_99.txt", package = "maftools")
scores.gis <- system.file("extdata", "scores.gistic", package = "maftools")

laml.gistic = readGistic(gisticAllLesionsFile = all.lesions, gisticAmpGenesFile = amp.genes, gisticDelGenesFile = del.genes, gisticScoresFile = scores.gis, isTCGA = TRUE)

#GISTIC object
laml.gistic

Similar to MAF objects, there are methods available to access slots of GISTIC object - getSampleSummary, getGeneSummary and getCytoBandSummary. Summarized results can be written to output files using function write.GisticSummary.

Visualizing gistic results.

There are three types of plots available to visualize gistic results.

genome plot

gisticChromPlot(gistic = laml.gistic, markBands = "all")

Bubble plot

gisticBubblePlot(gistic = laml.gistic)

oncoplot

This is similar to oncoplots except for copy number data. One can again sort the matrix according to annotations, if any. Below plot is the gistic results for LAML, sorted according to FAB classification. Plot shows that 7q deletions are virtually absent in M4 subtype where as it is widespread in other subtypes.

gisticOncoPlot(gistic = laml.gistic, clinicalData = getClinicalData(x = laml), clinicalFeatures = 'FAB_classification', sortByAnnotation = TRUE, top = 10)

Visualizing CBS segments

tcga.ab.009.seg <- system.file("extdata", "TCGA.AB.3009.hg19.seg.txt", package = "maftools")
plotCBSsegments(cbsFile = tcga.ab.009.seg)

Analysis

Somatic Interactions

Mutually exclusive or co-occurring set of genes can be detected using somaticInteractions function, which performs pair-wise Fisher's Exact test to detect such significant pair of genes. 

#exclusive/co-occurance event analysis on top 10 mutated genes. 
somaticInteractions(maf = laml, top = 25, pvalue = c(0.05, 0.1))

Detecting cancer driver genes based on positional clustering

maftools has a function oncodrive which identifies cancer genes (driver) from a given MAF. oncodrive is a based on algorithm oncodriveCLUST which was originally implemented in Python. Concept is based on the fact that most of the variants in cancer causing genes are enriched at few specific loci (aka hot-spots). This method takes advantage of such positions to identify cancer genes. If you use this function, please cite OncodriveCLUST article 7.

laml.sig = oncodrive(maf = laml, AACol = 'Protein_Change', minMut = 5, pvalMethod = 'zscore')

head(laml.sig)

We can plot the results using plotOncodrive.

plotOncodrive(res = laml.sig, fdrCutOff = 0.1, useFraction = TRUE, labelSize = 0.5)

plotOncodrive plots the results as scatter plot with size of the points proportional to the number of clusters found in the gene. X-axis shows number of mutations (or fraction of mutations) observed in these clusters. In the above example, IDH1 has a single cluster and all of the 18 mutations are accumulated within that cluster, giving it a cluster score of one. For details on oncodrive algorithm, please refer to OncodriveCLUST article 7.

Adding and summarizing pfam domains

maftools comes with the function pfamDomains, which adds pfam domain information to the amino acid changes. pfamDomain also summarizes amino acid changes according to the domains that are affected. This serves the purpose of knowing what domain in given cancer cohort, is most frequently affected. This function is inspired from Pfam annotation module from MuSic tool 8.

laml.pfam = pfamDomains(maf = laml, AACol = 'Protein_Change', top = 10)
#Protein summary (Printing first 7 columns for display convenience)
laml.pfam$proteinSummary[,1:7, with = FALSE]
#Domain summary (Printing first 3 columns for display convenience)
laml.pfam$domainSummary[,1:3, with = FALSE]

Survival analysis

Survival analysis is an essential part of cohort based sequencing projects. Function mafSurvive performs survival analysis and draws kaplan meier curve by grouping samples based on mutation status of user defined gene(s) or manually provided samples those make up a group. This function requires input data to contain Tumor_Sample_Barcode (make sure they match to those in MAF file), binary event (1/0) and time to event.

Our annotation data already contains survival information and in case you have survival data stored in a separate table provide them via argument clinicalData

Mutation in any given genes

#Survival analysis based on grouping of DNMT3A mutation status
mafSurvival(maf = laml, genes = 'DNMT3A', time = 'days_to_last_followup', Status = 'Overall_Survival_Status', isTCGA = TRUE)

Predict genesets associated with survival

Identify set of genes which results in poor survival

#Using top 20 mutated genes to identify a set of genes (of size 2) to predict poor prognostic groups
prog_geneset = survGroup(maf = laml, top = 20, geneSetSize = 2, time = "days_to_last_followup", Status = "Overall_Survival_Status", verbose = FALSE)

print(prog_geneset)

Above results show a combination (N = 2) of genes which are associated with poor survival (P < 0.05). We can draw KM curve for above results with the function mafSurvGroup

mafSurvGroup(maf = laml, geneSet = c("DNMT3A", "FLT3"), time = "days_to_last_followup", Status = "Overall_Survival_Status")

Comparing two cohorts (MAFs)

Cancers differ from each other in terms of their mutation pattern. We can compare two different cohorts to detect such differentially mutated genes. For example, recent article by Madan et. al 9, have shown that patients with relapsed APL (Acute Promyelocytic Leukemia) tends to have mutations in PML and RARA genes, which were absent during primary stage of the disease. This difference between two cohorts (in this case primary and relapse APL) can be detected using function mafComapre, which performs fisher test on all genes between two cohorts to detect differentially mutated genes.

#Primary APL MAF
primary.apl = system.file("extdata", "APL_primary.maf.gz", package = "maftools")
primary.apl = read.maf(maf = primary.apl)
#Relapse APL MAF
relapse.apl = system.file("extdata", "APL_relapse.maf.gz", package = "maftools")
relapse.apl = read.maf(maf = relapse.apl)

#Considering only genes which are mutated in at-least in 5 samples in one of the cohort to avoid bias due to genes mutated in single sample.
pt.vs.rt <- mafCompare(m1 = primary.apl, m2 = relapse.apl, m1Name = 'Primary', m2Name = 'Relapse', minMut = 5)
print(pt.vs.rt)

Forest plots

Above results show two genes PML and RARA which are highly mutated in Relapse APL compared to Primary APL. We can visualize these results as a forestplot.

forestPlot(mafCompareRes = pt.vs.rt, pVal = 0.1, color = c('royalblue', 'maroon'), geneFontSize = 0.8)

Co-onco plots

Another alternative way of displaying above results is by plotting two oncoplots side by side. coOncoplot function takes two maf objects and plots them side by side for better comparison.

genes = c("PML", "RARA", "RUNX1", "ARID1B", "FLT3")
coOncoplot(m1 = primary.apl, m2 = relapse.apl, m1Name = 'PrimaryAPL', m2Name = 'RelapseAPL', genes = genes, removeNonMutated = TRUE)

Co-bar plots

coBarplot(m1 = primary.apl, m2 = relapse.apl, m1Name = "Primary", m2Name = "Relapse")

Lollipop plot-2

Along with plots showing cohort wise differences, its also possible to show gene wise differences with lollipopPlot2 function.

lollipopPlot2(m1 = primary.apl, m2 = relapse.apl, gene = "PML", AACol1 = "amino_acid_change", AACol2 = "amino_acid_change", m1_name = "Primary", m2_name = "Relapse")

Clinical enrichment analysis

clinicalEnrichment is another function which takes any clinical feature associated with the samples and performs enrichment analysis. It performs various groupwise and pairwise comparisions to identify enriched mutations for every category within a clincila feature. Below is an example to identify mutations associated with FAB_classification.

fab.ce = clinicalEnrichment(maf = laml, clinicalFeature = 'FAB_classification')

#Results are returned as a list. Significant associations p-value < 0.05
fab.ce$groupwise_comparision[p_value < 0.05]

Above results shows IDH1 mutations are enriched in M1 subtype of leukemia compared to rest of the cohort. Similarly DNMT3A is in M5, RUNX1 is in M0, and so on. These are well known results and this function effectively recaptures them. One can use any sort of clincial feature to perform such an analysis. There is also a small function - plotEnrichmentResults which can be used to plot these results.

plotEnrichmentResults(enrich_res = fab.ce, pVal = 0.05, geneFontSize = 0.5, annoFontSize = 0.6)

Drug-Gene Interactions

drugInteractions function checks for drug–gene interactions and gene druggability information compiled from Drug Gene Interaction database. 

dgi = drugInteractions(maf = laml, fontSize = 0.75)

Above plot shows potential druggable gene categories along with upto top 5 genes involved in them. One can also extract information on drug-gene interactions. For example below is the results for known/reported drugs to interact with DNMT3A. 

dnmt3a.dgi = drugInteractions(genes = "DNMT3A", drugs = TRUE)
#Printing selected columns.
dnmt3a.dgi[,.(Gene, interaction_types, drug_name, drug_claim_name)]

Please cite DGIdb article if you find this function useful 10.

Disclaimer: Resources used in this function are intended for purely research purposes. It should not be used for emergencies or medical or professional advice.

Oncogenic Signaling Pathways

OncogenicPathways function checks for enrichment of known Oncogenic Signaling Pathways in TCGA cohorts 11.

OncogenicPathways(maf = laml)

Its also possible to visualize complete pathway.

PlotOncogenicPathways(maf = laml, pathways = "RTK-RAS")

Tumor suppressor genes are in red, and oncogenes are in blue font.

Tumor heterogeneity and MATH scores

Heterogeneity in tumor samples

Tumors are generally heterogeneous i.e, consist of multiple clones. This heterogeneity can be inferred by clustering variant allele frequencies. inferHeterogeneity function uses vaf information to cluster variants (using mclust), to infer clonality. By default, inferHeterogeneity function looks for column t_vaf containing vaf information. However, if the field name is different from t_vaf, we can manually specify it using argument vafCol. For example, in this case study vaf is stored under the field name i_TumorVAF_WU.

#Heterogeneity in sample TCGA.AB.2972
library("mclust")
tcga.ab.2972.het = inferHeterogeneity(maf = laml, tsb = 'TCGA-AB-2972', vafCol = 'i_TumorVAF_WU')
print(tcga.ab.2972.het$clusterMeans)
#Visualizing results
plotClusters(clusters = tcga.ab.2972.het)

Above figure shows clear separation of two clones clustered at mean variant allele frequencies of ~45% (major clone) and another minor clone at variant allele frequency of ~25%.

Although clustering of variant allele frequencies gives us a fair idea on heterogeneity, it is also possible to measure the extent of heterogeneity in terms of a numerical value. MATH score (mentioned as a subtitle in above plot) is a simple quantitative measure of intra-tumor heterogeneity, which calculates the width of the vaf distribution. Higher MATH scores are found to be associated with poor outcome. MATH score can also be used a proxy variable for survival analysis 11.

Ignoring variants in copy number altered regions

We can use copy number information to ignore variants located on copy-number altered regions. Copy number alterations results in abnormally high/low variant allele frequencies, which tends to affect clustering. Removing such variants improves clustering and density estimation while retaining biologically meaningful results. Copy number information can be provided as a segmented file generated from segmentation programmes, such as Circular Binary Segmentation from "DNACopy" Bioconductor package 6.

seg = system.file('extdata', 'TCGA.AB.3009.hg19.seg.txt', package = 'maftools')
tcga.ab.3009.het = inferHeterogeneity(maf = laml, tsb = 'TCGA-AB-3009', segFile = seg, vafCol = 'i_TumorVAF_WU')
#Visualizing results. Highlighting those variants on copynumber altered variants.
plotClusters(clusters = tcga.ab.3009.het, genes = 'CN_altered', showCNvars = TRUE)

Above figure shows two genes NF1 and SUZ12 with high VAF's, which is due to copy number alterations (deletion). Those two genes are ignored from analysis.

Mutational Signatures

Every cancer, as it progresses leaves a signature characterized by specific pattern of nucleotide substitutions. Alexandrov et.al have shown such mutational signatures, derived from over 7000 cancer samples 5. Such signatures can be extracted by decomposing matrix of nucleotide substitutions, classified into 96 substitution classes based on immediate bases surrounding the mutated base. Extracted signatures can also be compared to those validated signatures.

First step in signature analysis is to obtain the adjacent bases surrounding the mutated base and form a mutation matrix. NOTE: Earlier versions of maftools required a fasta file as an input. But starting from 1.8.0, BSgenome objects are used for faster sequence extraction.

#Requires BSgenome object
library(BSgenome.Hsapiens.UCSC.hg19, quietly = TRUE)
laml.tnm = trinucleotideMatrix(maf = laml, prefix = 'chr', add = TRUE, ref_genome = "BSgenome.Hsapiens.UCSC.hg19")

Above function performs two steps:

APOBEC Enrichment estimation.

APOBEC induced mutations are more frequent in solid tumors and are mainly associated with C>T transition events occurring in TCW motif. APOBEC enrichment scores in the above command are estimated using the method described by Roberts et al 13. Briefly, enrichment of C>T mutations occurring within TCW motif over all of the C>T mutations in a given sample is compared to background Cytosines and TCWs occurring within 20bp of mutated bases.

$$\frac{n_{tCw} * background_C}{n_C * background_{TCW}}$$

One-sided fishers exact test is also performed to statistically evaluate the enrichment score, as described in original study by Roberts et al.

Differences between APOBEC enriched and non-enriched samples

We can also analyze the differences in mutational patterns between APOBEC enriched and non-APOBEC enriched samples. plotApobecDiff is a function which takes APOBEC enrichment scores estimated by trinucleotideMatrix and classifies samples into APOBEC enriched and non-APOBEC enriched. Once stratified, it compares these two groups to identify differentially altered genes.

Note that, LAML with no APOBEC enrichments, is not an ideal cohort for this sort of analysis and hence below plot is only for demonstration purpose.

plotApobecDiff(tnm = laml.tnm, maf = laml, pVal = 0.2)

Signature analysis

Signature analysis includes following steps.

  1. estimateSignatures - which runs NMF on a range of values and measures the goodness of fit - in terms of Cophenetic correlation.
  2. plotCophenetic - which draws an elblow plot and helps you to decide optimal number of signatures. Best possible signature is the value at which Cophenetic correlation drops significantly.
  3. extractSignatures - uses non-negative matrix factorization to decompose the matrix into n signatures. n is chosen based on the above two steps. In case if you already have a good estimate of n, you can skip above two steps.
  4. compareSignatures - extracted signatures from above step can be compared to known signatures11 from COSMIC database, and cosine similarity is calculated to identify best match.
  5. plotSignatures - plots signatures
par(mar = c(2, 2, 2, 1))
plot(NA, xlim = c(1, 10), ylim = c(0, 30), frame.plot = FALSE, axes = FALSE, xlab = NA, ylab = NA)
rect(xleft = 3, ybottom = 28, xright = 7, ytop = 30, col = grDevices::adjustcolor("gray70", alpha.f = 0.6), lwd = 1.2, border = "maroon")
text(x = 5, y = 29, labels = "MAF", font = 2)
arrows(x0 = 5, y0 = 28, x1 = 5, y1 = 26, length = 0.1, lwd = 2)
text(x = 5, y = 25, labels = "trinucleotideMatrix()", font = 3)
arrows(x0 = 5, y0 = 24, x1 = 5, y1 = 21, length = 0.1, lwd = 2)
text(x = 5, y = 20, labels = "estimateSignatures()", font = 3)
arrows(x0 = 5, y0 = 19, x1 = 5, y1 = 16, length = 0.1, lwd = 2)
text(x = 5, y = 15, labels = "plotCophenetic()", font = 3)
arrows(x0 = 5, y0 = 14, x1 = 5, y1 = 11, length = 0.1, lwd = 2)
text(x = 5, y = 10, labels = "extractSignatures()", font = 3)
arrows(x0 = 5, y0 = 9, x1 = 5, y1 = 6, length = 0.1, lwd = 2)
text(x = 5, y = 5, labels = "compareSignatures()", font = 3)
arrows(x0 = 5, y0 = 4, x1 = 5, y1 = 1, length = 0.1, lwd = 2)
text(x = 5, y = 0, labels = "plotSignatures()", font = 3)

Note: In previous versions, extractSignatures used to take care of above steps automatically. After versions 2.2.0, main function is split inot above 5 stpes for user flexibility.

library('NMF')
laml.sign = estimateSignatures(mat = laml.tnm, nTry = 6)

#Run main function with maximum 6 signatures. 
library('NMF')
laml.sign = estimateSignatures(mat = laml.tnm, nTry = 6, pConstant = 0.1, plotBestFitRes = FALSE, parallel = 2)

Draw elbow plot to visualize and decide optimal number of signatures from above results.

plotCophenetic(res = laml.sign)

Best possible value is the one at which the correlation value on the y-axis drops significantly. In this case it appears to be at n = 3. LAML is not an ideal example for signature analysis with its low mutation rate, but for solid tumors with higher mutation burden one could expect more signatures, provided sufficient number of samples.

Once n is estimated, we can run the main function.

laml.sig = extractSignatures(mat = laml.tnm, n = 3)

laml.sig = extractSignatures(mat = laml.tnm, n = 3, pConstant = 0.1,  parallel = 2)

Compare detected signatures to COSMIC Legacy or SBS signature database.

#Compate against original 30 signatures 
laml.og30.cosm = compareSignatures(nmfRes = laml.sig, sig_db = "legacy")
#Compate against updated version3 60 signatures 
laml.v3.cosm = compareSignatures(nmfRes = laml.sig, sig_db = "SBS")

compareSignatures returns full table of cosine similarities against COSMIC signatures, which can be further analysed. Below plot shows comparison of similarities of detected signatures against validated signatures.

library('pheatmap')
pheatmap::pheatmap(mat = laml.og30.cosm$cosine_similarities, cluster_rows = FALSE, main = "cosine similarity against validated signatures")

Finally plot signatures

maftools::plotSignatures(nmfRes = laml.sig, title_size = 1.2, sig_db = "SBS")

If you fancy 3D barpots, you can install barplot3d package and visualize the results with legoplot3d function.

library("barplot3d")
#Visualize first signature
sig1 = laml.sig$signatures[,1]
barplot3d::legoplot3d(contextdata = sig1, labels = FALSE, scalexy = 0.01, sixcolors = "sanger", alpha = 0.5)

NOTE:

  1. Should you receive an error while running extractSignatures complaining none of the packages are loaded, please manually load the NMF library and re-run.

  2. If either extractSignatures or estimateSignatures stops in between, its possibly due to low mutation counts in the matrix. In that case rerun the functions with pConstant argument set to small positive value (e.g, 0.1).

Variant Annotations

Converting annovar output to MAF

Annovar is one of the most widely used Variant Annotation tool in Genomics 17. Annovar output is generally in a tabular format with various annotation columns. This function converts such annovar output files into MAF. This function requires that annovar was run with gene based annotation as a first operation, before including any filter or region based annotations.

e.g, 

table_annovar.pl example/ex1.avinput humandb/ -buildver hg19 -out myanno -remove -protocol (refGene),cytoBand,dbnsfp30a -operation (g),r,f -nastring NA

annovarToMaf mainly uses gene based annotations for processing, rest of the annotation columns from input file will be attached to the end of the resulting MAF.

As an example we will annotate the same file which was used above to run oncotate function. We will annotate it using annovar with the following command. For simplicity, here we are including only gene based annotations but one can include as many annotations as they wish. But make sure the fist operation is always gene based annotation.

```{bash, eval = F} $perl table_annovar.pl variants.tsv ~/path/to/humandb/ -buildver hg19 -out variants --otherinfo -remove -protocol ensGene -operation g -nastring NA

Output generated is stored as a part of this package. We can convert this annovar output into MAF using `annovarToMaf`. 

```r
var.annovar = system.file("extdata", "variants.hg19_multianno.txt", package = "maftools")
var.annovar.maf = annovarToMaf(annovar = var.annovar, Center = 'CSI-NUS', refBuild = 'hg19', 
                               tsbCol = 'Tumor_Sample_Barcode', table = 'ensGene')

Annovar, when used with Ensemble as a gene annotation source, uses ensemble gene IDs as Gene names. In that case, use annovarToMaf with argument table set to ensGene which converts ensemble gene IDs into HGNC symbols.

Converting ICGC Simple Somatic Mutation Format to MAF

Just like TCGA, International Cancer Genome Consortium ICGC also makes its data publicly available. But the data are stored in Simpale Somatic Mutation Format, which is similar to MAF format in its structure. However field names and classification of variants is different from that of MAF. icgcSimpleMutationToMAF is a function which reads ICGC data and converts them to MAF.

#Read sample ICGC data for ESCA
esca.icgc <- system.file("extdata", "simple_somatic_mutation.open.ESCA-CN.sample.tsv.gz", package = "maftools")
esca.maf <- icgcSimpleMutationToMAF(icgc = esca.icgc, addHugoSymbol = TRUE)
#Printing first 16 columns for display convenience.
print(esca.maf[1:5,1:16, with = FALSE])

Note that by default Simple Somatic Mutation format contains all affected transcripts of a variant resulting in multiple entries of the same variant in same sample. It is hard to choose a single affected transcript based on annotations alone and by default this program removes repeated variants as duplicated entries. If you wish to keep all of them, set removeDuplicatedVariants to FALSE. Another option is to convert input file to MAF by removing duplicates and then use scripts like vcf2maf to re-annotate and prioritize affected transcripts.

Prepare MAF file for MutSigCV analysis

MutSig/MutSigCV is most widely used program for detecting driver genes 18. However, we have observed that covariates files (gene.covariates.txt and exome_full192.coverage.txt) which are bundled with MutSig have non-standard gene names (non Hugo_Symbols). This discrepancy between Hugo_Symbols in MAF and non-Hugo_symbols in covariates file causes MutSig program to ignore such genes. For example, KMT2D - a well known driver gene in Esophageal Carcinoma is represented as MLL2 in MutSig covariates. This causes KMT2D to be ignored from analysis and is represented as an insignificant gene in MutSig results. This function attempts to correct such gene symbols with a manually curated list of gene names compatible with MutSig covariates list.

laml.mutsig.corrected = prepareMutSig(maf = laml)
# Converting gene names for 1 variants from 1 genes
#    Hugo_Symbol MutSig_Synonym N
# 1:    ARHGAP35          GRLF1 1
# Original symbols are preserved under column OG_Hugo_Symbol.

Set operations

Subsetting MAF

We can also subset MAF using function subsetMaf

#Extract data for samples 'TCGA.AB.3009' and 'TCGA.AB.2933'  (Printing just 5 rows for display convenience)
subsetMaf(maf = laml, tsb = c('TCGA-AB-3009', 'TCGA-AB-2933'), mafObj = FALSE)[1:5]
##Same as above but return output as an MAF object (Default behaviour)
subsetMaf(maf = laml, tsb = c('TCGA-AB-3009', 'TCGA-AB-2933'))

Specifying queries and controlling output fields.

#Select all Splice_Site mutations from DNMT3A and NPM1
subsetMaf(maf = laml, genes = c('DNMT3A', 'NPM1'), mafObj = FALSE,query = "Variant_Classification == 'Splice_Site'")

#Same as above but include only 'i_transcript_name' column in the output
subsetMaf(maf = laml, genes = c('DNMT3A', 'NPM1'), mafObj = FALSE, query = "Variant_Classification == 'Splice_Site'", fields = 'i_transcript_name')

Subsetting by clinical data

Use clinQuery argument in subsetMaf to select samples of interest based on their clinical features.

#Select all samples with FAB clasification M4 in clinical data 
laml_m4 = subsetMaf(maf = laml, clinQuery = "FAB_classification %in% 'M4'")

Pre-compiled TCGA MAF objects

There is also an R data package containing pre-compiled TCGA MAF objects from TCGA firehose and TCGA MC3 projects, particularly helpful for those working with TCGA mutation data. Every dataset is stored as an MAF object containing somatic mutations along with clinical information. Due to Bioconductor package size limits and other difficulties, this was not submitted to Bioconductor. However, you can still download TCGAmutations package from GitHub. 

BiocManager::install(pkgs = "PoisonAlien/TCGAmutations")

Useful links

| File formats | Data Portals | Annotation tools | |:------------------------------------------------------------------------------------------------------------------:|:-----------------------------------------------------------------------------------------------:|:---------------------------------------------------------------------------------------------:| | Mutation Annotation Format | TCGA | vcf2maf - for converting your VCF files to MAF | | Variant Call Format | ICGC | Ensembl Variant Effect Predictor VEP | | ICGC Simple Somatic Mutation Format | Broad Firehose | Annovar | | | cBioPortal | Funcotator | | | CIViC - Clinical interpretation of variants in cancer | | | | DGIdb - Information on drug-gene interactions and the druggable genome | |

Similar packages/tools

Below are some more useful software packages for somatic variant analysis (not necessarily similar to maftools)

References

  1. Cancer Genome Atlas Research, N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med 368, 2059-74 (2013).
  2. Mermel, C.H. et al. GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers. Genome Biol 12, R41 (2011).
  3. Olshen, A.B., Venkatraman, E.S., Lucito, R. & Wigler, M. Circular binary segmentation for the analysis of array-based DNA copy number data. Biostatistics 5, 557-72 (2004).
  4. Alexandrov, L.B. et al. Signatures of mutational processes in human cancer. Nature 500, 415-21 (2013).
  5. Tamborero, D., Gonzalez-Perez, A. & Lopez-Bigas, N. OncodriveCLUST: exploiting the positional clustering of somatic mutations to identify cancer genes. Bioinformatics 29, 2238-44 (2013).
  6. Dees, N.D. et al. MuSiC: identifying mutational significance in cancer genomes. Genome Res 22, 1589-98 (2012).
  7. Lawrence MS, Stojanov P, Mermel CH, Robinson JT, Garraway LA, Golub TR, Meyerson M, Gabriel SB, Lander ES, Getz G: Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 2014, 505:495-501.
  8. Griffith, M., Griffith, O. L., Coffman, A. C., Weible, J. V., McMichael, J. F., Spies, N. C., … Wilson, R. K. (2013). DGIdb - Mining the druggable genome. Nature Methods, 10(12), 1209–1210. http://doi.org/10.1038/nmeth.2689
  9. Sanchez-Vega F, Mina M, Armenia J, Chatila WK, Luna A, La KC, Dimitriadoy S, Liu DL, Kantheti HS, Saghafinia S et al. 2018. Oncogenic Signaling Pathways in The Cancer Genome Atlas. Cell 173: 321-337 e310
  10. Madan, V. et al. Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia. Leukemia 30, 1672-81 (2016).
  11. Mroz, E.A. & Rocco, J.W. MATH, a novel measure of intratumor genetic heterogeneity, is high in poor-outcome classes of head and neck squamous cell carcinoma. Oral Oncol 49, 211-5 (2013).
  12. Roberts SA, Lawrence MS, Klimczak LJ, et al. An APOBEC Cytidine Deaminase Mutagenesis Pattern is Widespread in Human Cancers. Nature genetics. 2013;45(9):970-976.
  13. Gaujoux, R. & Seoighe, C. A flexible R package for nonnegative matrix factorization. BMC Bioinformatics 11, 367 (2010).
  14. Welch, J.S. et al. The origin and evolution of mutations in acute myeloid leukemia. Cell 150, 264-78 (2012).
  15. Ramos, A.H. et al. Oncotator: cancer variant annotation tool. Hum Mutat 36, E2423-9 (2015).
  16. Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38, e164 (2010).
  17. Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, Carter SL, Stewart C, Mermel CH, Roberts SA, et al: Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 2013, 499:214-218.

Session Info

sessionInfo()

Support and acknowledgements

Github

If you have any issues, bug reports or feature requests please feel free to raise an issue on GitHub page.

Acknowledgements



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