Build OncoPrint and co-mutation matrix plots from somatic-variant cohorts using ComplexHeatmap, maftools, and comut.py with alteration-type stacking, sample ordering by mutational burden, mutual-exclusivity overlays, and clinical annotation tracks. Use when visualizing per-sample mutation patterns across recurrent driver genes, comparing alteration classes, or identifying mutually-exclusive / co-occurring driver pairs.
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Build OncoPrint and co-mutation matrix plots from somatic-variant cohorts using ComplexHeatmap, maftools, and comut.py with alteration-type stacking, sample ordering by mutational burden, mutual-exclusivity overlays, and clinical annotation tracks. Use when visualizing per-sample mutation patterns across recurrent driver genes, comparing alteration classes, or identifying mutually-exclusive / co-occurring driver pairs.
Before using code patterns, verify installed versions match. If versions differ:
Python: pip show <package> then help(module.function) to check signatures
R: packageVersion('<pkg>') then ?function_name
If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.
OncoPrint and Mutation Matrix Plots
"Plot mutations across a cohort" -> Render a gene-by-sample matrix where each cell stacks colored rectangles encoding alteration class (missense, truncating, splice, copy-gain, copy-loss, fusion). Sort samples by burden, optionally split by clinical group, and overlay co-mutation / mutual-exclusivity annotations. OncoPrint (Cerami 2012 2:401; canonical at cBioPortal) is the genre-defining visualization.
The Single Most Important Modern Insight -- Cell Stacking Encodes Multiple Alterations Per Cell
OncoPrint differs from a generic heatmap because each cell can encode multiple alterations simultaneously through stacked rectangles. A patient with both a missense and a copy-gain in TP53 shows one cell with two overlapping colored rectangles (e.g., green diamond inside red square). This stacking is the whole point — it preserves the multi-modal alteration landscape that flattening to a single category destroys.
In ComplexHeatmap's oncoPrint, the alter_fun argument is the rendering specification: a named list of functions, one per alteration class, each drawing its rectangle inside the cell. Get this right and the figure works; get it wrong and overlapping alterations are invisible.
Decision Tree by Cohort and Question
Question
Sort by
Display
Which genes are most altered?
Gene frequency (default)
Bar above samples (sample TMB); bar right of genes (gene frequency)
Per-patient burden patterns
Sample burden
TMB bar on top; sample-name labels
Subtype-driver enrichment
Clinical group then burden
column_split by group; per-group frequency right bar
Goal: Render a cohort mutation matrix with stacked alteration-class encoding, sample annotations, and a sample-sorted, gene-frequency-ranked layout.
Approach: Convert the MAF/variant table to a gene-by-sample matrix of ;-delimited alteration strings; define alter_fun rendering one rectangle per class; pass to oncoPrint() with column annotations.
library(ComplexHeatmap)
library(circlize)# Input: matrix where each cell is a string like 'Missense;Amp' or '' for no alteration# Rows = genes; columns = samples# Color per alteration class
col <-c('Missense'='#56B4E9','Truncating'='#000000','Splice'='#CC79A7','Amp'='#D55E00','HomDel'='#0072B2','Fusion'='#009E73')# alter_fun -- one function per class, each drawing inside the cell
alter_fun <-list(
background =function(x, y, w, h)
grid.rect(x, y, w - unit(0.5,'mm'), h - unit(0.5,'mm'),
gp = gpar(fill ='#EEEEEE', col =NA)),
Amp =function(x, y, w, h)
grid.rect(x, y, w - unit(0.5,'mm'), h - unit(0.5,'mm'),
gp = gpar(fill = col['Amp'], col =NA)),
HomDel =function(x, y, w, h)
grid.rect(x, y, w - unit(0.5,'mm'), h - unit(0.5,'mm'),
gp = gpar(fill = col['HomDel'], col =NA)),
Missense =function(x, y, w, h)
grid.rect(x, y, w - unit(0.5,'mm'), h *0.5,
gp = gpar(fill = col['Missense'], col =NA)),
Truncating =function(x, y, w, h)
grid.rect(x, y, w - unit(0.5,'mm'), h *0.33,
gp = gpar(fill = col['Truncating'], col =NA)),
Splice =function(x, y, w, h)
grid.rect(x, y, w - unit(0.5,'mm'), h *0.25,
gp = gpar(fill = col['Splice'], col =NA)),
Fusion =function(x, y, w, h)
grid.points(x, y, pch =17, size = unit(2,'mm'),
gp = gpar(col = col['Fusion'])))# Clinical column annotation
ha_clin <- HeatmapAnnotation(
Subtype = clinical$subtype,
Stage = clinical$stage,
col =list(Subtype =c(Luminal='#0072B2', Basal='#D55E00', HER2='#009E73'),
Stage =c(I='#FFFFCC', II='#FED976', III='#FD8D3C', IV='#BD0026')))
oncoPrint(mat,
alter_fun = alter_fun,
col = col,
top_annotation = ha_clin,
column_title ='TCGA-BRCA mutation landscape',
row_names_gp = gpar(fontsize =8),
pct_gp = gpar(fontsize =7),
show_pct =TRUE,
remove_empty_columns =FALSE,
remove_empty_rows =FALSE)
maftools::oncoplot -- Faster Onboarding
For TCGA-style MAF files, maftools::oncoplot is the lower-friction option:
maftools defaults handle alteration-class colors, sample sorting, and percentage bars automatically. Customization is more limited than ComplexHeatmap.
Mutual Exclusivity and Co-Occurrence
# maftools provides somaticInteractions
si <- somaticInteractions(maf = maf, top =20,
pvalue =c(0.05,0.01),
fontSize =0.7)# Plot returns a matrix of -log10(p) with sign by direction (+ co-occur, - mutex)
Mutual-exclusivity testing on small cohorts (N < 50) is underpowered; reported "significant" mutex on n=20 with 2 mutations each is uninterpretable. Aggregate to larger cohorts (TCGA + ICGC pan-cancer) or report effect size with CI rather than p-value.
Fisher exact vs DISCOVER: standard 2x2 Fisher tests sample-mutation pairs, ignoring per-gene mutation rate background. DISCOVER (Canisius 2016 Genome Biol 17:261) models per-tumor mutation probability and is preferred for pan-cancer analyses where mutation rate varies 100× across samples.
Trigger: Reducing each cell to a single most-severe alteration, losing the stack.
Mechanism: Loses the multi-alteration biology (e.g., MYC amp + missense in TP53).
Symptom: OncoPrint looks like a simple heatmap; co-occurring multi-class events invisible.
Fix: Build the cell as ;-separated alteration string; define alter_fun for each class.
Sample sort by gene 1 frequency only
Trigger: Default oncoPrint sorts samples by altered-gene-1 status; weakens the "memo sort" pattern.
Mechanism: True OncoPrint uses memoSort (Cerami 2012) which sorts by the binary altered-or-not pattern across the top genes.
Symptom: Samples with the same alteration profile are not adjacent; "staircase" pattern lost.
Fix: ComplexHeatmap oncoPrint uses memoSort by default; do NOT override column_order unless intentional.
Showing only mutated samples (remove_empty_columns = TRUE)
Trigger: Default in some implementations.
Mechanism: Drops samples with no mutations in the displayed genes — but those samples ARE part of the cohort.
Symptom: Sample count differs from cohort N; denominator-based percentages wrong.
Fix:remove_empty_columns = FALSE to preserve all samples; percentages now reflect true cohort fraction.
Hypermutators dominate visual
Trigger: Cohort with 1-2 POLE-mutant or MSI-H samples; TMB bar saturates.
Mechanism: Hypermutator TMB is 10-100× the typical sample.
Symptom: All other samples' TMB bars are invisible; one column dominates.
Fix: Log-transform the TMB annotation: anno_barplot(log10(tmb + 1)); OR cap with ylim.
Mutex/co-occurrence p-values overinterpreted on small cohorts
Trigger: Fisher exact test on N < 50 with low mutation counts.
Mechanism: With 2 mutations vs 3 mutations in 20 samples, all p-values are dominated by noise.
Symptom: "Significant mutex" claim from a tiny pilot.
Fix: Aggregate to ≥100 samples for credible mutex; use DISCOVER (Canisius 2016) instead of Fisher when mutation rate varies 100× across samples.
Small-Cohort Regime (N = 20-50)
For rare-cancer cohorts where N < 50, the standard OncoPrint + Fisher mutex pipeline is statistically uninterpretable:
Action
What to do
Report per-gene frequencies
Use exact-binomial CI (Clopper-Pearson via binom.test) — Wald CI is invalid at low frequency
Do NOT report mutex p-values
Fisher exact on 2x2 with cell counts ≤ 5 has no power; the "significant" mutex finding is noise
Hypothesis generation only
Pool with TCGA Pan-Cancer + ICGC for credible mutex; treat the cohort as the replication not the discovery
Co-occurrence reporting
OR with Haldane-Anscombe 0.5 correction for zero cells; report alongside cohort N
Show the OncoPrint for visual transparency, but the per-gene-frequency table (with exact-binomial CIs) is the load-bearing scientific output, not the mutex test.
Reconciliation: When Implementations Differ
Pattern
Cause
Action
ComplexHeatmap and maftools show different sample orders
Different memoSort defaults
Specify sortByAnnotation explicitly; report sort criterion in caption
Percentage labels differ
remove_empty_columns = TRUE vs FALSE
Document denominator (cohort-N vs altered-N)
Some alterations missing from a sample
Filtering: silent SNVs, low VAF
Document filtering criteria upstream
Quantitative Thresholds
Threshold
Value
Source
Cohort N for valid mutex
≥100 (pan-cancer); ≥50 (single-cohort with effect-size focus)
Common practice
Display top genes
10-25 in single panel
More creates visual clutter
Sample N for OncoPrint
50-1000 (above: switch to summary panel)
Visualization practical
Common Errors
Error / symptom
Cause
Solution
Co-occurring multi-class events invisible
Single-class flattening
Use ;-separated cells + alter_fun list
Sample order doesn't show staircase
column_order override
Trust default memoSort
Sample count differs from cohort
remove_empty_columns = TRUE
Set to FALSE
TMB bar dominated by 1-2 samples
Hypermutators on linear scale
log10 + 1 transform
Mutex p-values on N=20
Underpowered
Aggregate cohorts; use DISCOVER
Gene frequency right-bar mismatches percentages
Denominator definition
Document cohort-N vs altered-N
References
Canisius S, Martens JWM, Wessels LFA. 2016. A novel independence test for somatic alterations in cancer shows that biology drives mutual exclusivity but chance explains most co-occurrence. Genome Biol 17:261.
Cerami E, Gao J, Dogrusoz U, et al. 2012. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2(5):401-404.
Gao J, Aksoy BA, Dogrusoz U, et al. 2013. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 6(269):pl1.
Gu Z, Eils R, Schlesner M. 2016. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32(18):2847-2849.
Mayakonda A, Lin DC, Assenov Y, Plass C, Koeffler HP. 2018. Maftools: efficient and comprehensive analysis of somatic variants in cancer. Genome Res 28(11):1747-1756.
