Mutation Analysis (MutSig v1.5)
Colorectal Adenocarcinoma (Primary solid tumor)
02 April 2015  |  analyses__2015_04_02
Maintainer Information
Citation Information
doi:10.7908/C15H7F8D
Maintained by David Heiman (Broad Institute)
Cite as Broad Institute TCGA Genome Data Analysis Center (2015): Mutation Analysis (MutSig v1.5). Broad Institute of MIT and Harvard. doi:10.7908/C15H7F8D
Overview
Introduction

This report serves to describe the mutational landscape and properties of a given individual set, as well as rank genes and genesets according to mutational significance. MutSig v1.5 was used to generate the results found in this report.

  • Working with individual set: COADREAD-TP

  • Number of patients in set: 488

Input

The input for this pipeline is a set of individuals with the following files associated for each:

  1. An annotated .maf file describing the mutations called for the respective individual, and their properties.

  2. A .wig file that contains information about the coverage of the sample.

Summary

  • MAF used for this analysis:COADREAD-TP.final_analysis_set.maf

  • Blacklist used for this analysis: pancan_mutation_blacklist.v14.hg19.txt

  • Significantly mutated genes (q ≤ 0.1): 181

  • Mutations seen in COSMIC: 9998

  • Significantly mutated genes in COSMIC territory: 1678

  • Significantly mutated genesets: 3

  • Significantly mutated genesets: (excluding sig. mutated genes):0

Mutation Preprocessing

  • Read 140 MAFs of type "Broad"

  • Read 266 MAFs of type "Baylor-Illumina"

  • Read 87 MAFs of type "Baylor-SOLiD"

  • Total number of mutations in input MAFs: 241105

  • After removing 2 blacklisted mutations: 241103

  • After removing 72 noncoding mutations: 241031

  • After collapsing adjacent/redundant mutations: 230471

Mutation Filtering

  • Number of mutations before filtering: 230471

  • After removing 127 mutations outside patient set: 230344

  • After removing 6942 mutations outside gene set: 223402

  • After removing 1207 mutations outside category set: 222195

  • After removing 4238 "impossible" mutations in

  • gene-patient-category bins of zero coverage: 209681

Results
Breakdown of Mutations by Type

Table 1.  Get Full Table Table representing breakdown of mutations by type.

type count
De_novo_Start_InFrame 20
De_novo_Start_OutOfFrame 155
Frame_Shift_Del 13132
Frame_Shift_Ins 3998
In_Frame_Del 1723
In_Frame_Ins 596
Missense_Mutation 136150
Nonsense_Mutation 10627
Nonstop_Mutation 45
Silent 53959
Splice_Site 1788
Translation_Start_Site 2
Total 222195
Breakdown of Mutation Rates by Category Type

Table 2.  Get Full Table A breakdown of mutation rates per category discovered for this individual set.

category n N rate rate_per_mb relative_rate exp_ns_s_ratio
*CpG->T 45076 647938249 7e-05 70 5.5 2.2
A->G 27228 6470281486 4.2e-06 4.2 0.33 2.3
*Cp(A/C/T)->T 19486 5814253842 3.4e-06 3.4 0.26 1.7
transver 41220 12932473577 3.2e-06 3.2 0.25 5.1
indel+null 30618 12932473577 2.4e-06 2.4 0.19 NaN
double_null 1028 12932473577 7.9e-08 0.079 0.0062 NaN
Total 164656 12932473577 0.000013 13 1 3.5
Target Coverage for Each Individual

The x axis represents the samples. The y axis represents the exons, one row per exon, and they are sorted by average coverage across samples. For exons with exactly the same average coverage, they are sorted next by the %GC of the exon. (The secondary sort is especially useful for the zero-coverage exons at the bottom). If the figure is unpopulated, then full coverage is assumed (e.g. MutSig CV doesn't use WIGs and assumes full coverage).

Figure 1. 

Distribution of Mutation Counts, Coverage, and Mutation Rates Across Samples

Figure 2.  Patients counts and rates file used to generate this plot: COADREAD-TP.patients.counts_and_rates.txt

Lego Plots

The mutation spectrum is depicted in the lego plots below in which the 96 possible mutation types are subdivided into six large blocks, color-coded to reflect the base substitution type. Each large block is further subdivided into the 16 possible pairs of 5' and 3' neighbors, as listed in the 4x4 trinucleotide context legend. The height of each block corresponds to the mutation frequency for that kind of mutation (counts of mutations normalized by the base coverage in a given bin). The shape of the spectrum is a signature for dominant mutational mechanisms in different tumor types.

Figure 3.  Get High-res Image SNV Mutation rate lego plot for entire set. Each bin is normalized by base coverage for that bin. Colors represent the six SNV types on the upper right. The three-base context for each mutation is labeled in the 4x4 legend on the lower right. The fractional breakdown of SNV counts is shown in the pie chart on the upper left. If this figure is blank, not enough information was provided in the MAF to generate it.

Figure 4.  Get High-res Image SNV Mutation rate lego plots for 4 slices of mutation allele fraction (0<=AF<0.1, 0.1<=AF<0.25, 0.25<=AF<0.5, & 0.5<=AF) . The color code and three-base context legends are the same as the previous figure. If this figure is blank, not enough information was provided in the MAF to generate it.

CoMut Plot

Figure 5.  Get High-res Image The matrix in the center of the figure represents individual mutations in patient samples, color-coded by type of mutation, for the significantly mutated genes. The rate of synonymous and non-synonymous mutations is displayed at the top of the matrix. The barplot on the left of the matrix shows the number of mutations in each gene. The percentages represent the fraction of tumors with at least one mutation in the specified gene. The barplot to the right of the matrix displays the q-values for the most significantly mutated genes. The purple boxplots below the matrix (only displayed if required columns are present in the provided MAF) represent the distributions of allelic fractions observed in each sample. The plot at the bottom represents the base substitution distribution of individual samples, using the same categories that were used to calculate significance.

Significantly Mutated Genes

Column Descriptions:

  • N = number of sequenced bases in this gene across the individual set

  • n = number of (nonsilent) mutations in this gene across the individual set

  • npat = number of patients (individuals) with at least one nonsilent mutation

  • nsite = number of unique sites having a non-silent mutation

  • nsil = number of silent mutations in this gene across the individual set

  • n1 = number of nonsilent mutations of type: *CpG->T

  • n2 = number of nonsilent mutations of type: A->G

  • n3 = number of nonsilent mutations of type: *Cp(A/C/T)->T

  • n4 = number of nonsilent mutations of type: transver

  • n5 = number of nonsilent mutations of type: indel+null

  • n6 = number of nonsilent mutations of type: double_null

  • p_ns_s = p-value for the observed nonsilent/silent ratio being elevated in this gene

  • p = p-value (overall)

  • q = q-value, False Discovery Rate (Benjamini-Hochberg procedure)

Table 3.  Get Full Table A Ranked List of Significantly Mutated Genes. Number of significant genes found: 181. Number of genes displayed: 35. Click on a gene name to display its stick figure depicting the distribution of mutations and mutation types across the chosen gene (this feature may not be available for all significant genes).

rank gene description N n npat nsite nsil n1 n2 n3 n4 n5 n6 p_ns_s p q
1 CRIPAK cysteine-rich PAK1 inhibitor 352654 71 60 27 7 5 11 2 8 37 8 0.14 <1.00e-15 <3.92e-12
2 MYL1 myosin, light chain 1, alkali; skeletal, fast 296955 36 36 5 6 1 1 0 1 32 1 1 <1.00e-15 <3.92e-12
3 HLA-DQA1 major histocompatibility complex, class II, DQ alpha 1 197238 33 30 5 0 1 0 1 0 23 8 0.34 <1.00e-15 <3.92e-12
4 DDHD1 DDHD domain containing 1 1111723 51 50 5 1 2 0 0 1 48 0 0.56 1.11e-15 3.92e-12
5 RBM38 RNA binding motif protein 38 95557 42 43 5 2 2 0 0 2 38 0 0.52 1.22e-15 3.92e-12
6 ZNF516 zinc finger protein 516 540665 36 37 16 3 7 1 3 0 25 0 0.19 1.33e-15 3.92e-12
7 NEFH neurofilament, heavy polypeptide 200kDa 869213 111 109 21 4 1 1 1 3 105 0 0.91 1.55e-15 3.92e-12
8 KRAS v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog 341646 247 232 42 21 2 22 125 95 3 0 4.1e-11 2.78e-15 5.05e-12
9 ORAI1 ORAI calcium release-activated calcium modulator 1 275118 44 44 4 0 2 0 0 1 41 0 0.28 3.55e-15 5.05e-12
10 CDH1 cadherin 1, type 1, E-cadherin (epithelial) 1274697 99 86 72 21 10 41 13 12 22 1 0.0013 4.11e-15 5.05e-12
11 EYS eyes shut homolog (Drosophila) 1410067 73 52 63 8 4 1 12 29 25 2 0.067 4.33e-15 5.05e-12
12 TCF7L2 transcription factor 7-like 2 (T-cell specific, HMG-box) 889185 58 54 42 6 12 3 4 11 28 0 0.0039 4.44e-15 5.05e-12
13 KRTAP10-7 keratin associated protein 10-7 330364 44 45 5 1 0 1 0 1 42 0 0.85 4.66e-15 5.05e-12
14 DEFB126 defensin, beta 126 164975 39 37 5 0 2 0 1 1 35 0 0.2 4.66e-15 5.05e-12
15 CASP5 caspase 5, apoptosis-related cysteine peptidase 637599 56 55 15 3 3 5 1 4 43 0 0.29 4.77e-15 5.05e-12
16 KRTAP4-1 keratin associated protein 4-1 117393 47 45 3 0 3 0 0 0 44 0 1 4.77e-15 5.05e-12
17 GPRIN2 G protein regulated inducer of neurite outgrowth 2 298630 40 38 7 4 2 0 0 3 35 0 0.9 5.33e-15 5.05e-12
18 IFI27 interferon, alpha-inducible protein 27 163853 19 19 3 1 1 1 1 0 16 0 0.55 5.55e-15 5.05e-12
19 MUC4 mucin 4, cell surface associated 1493015 260 158 85 31 8 14 5 17 209 7 1 5.77e-15 5.05e-12
20 TP53 tumor protein p53 604338 435 321 222 78 130 92 48 74 87 4 1.7e-08 5.88e-15 5.05e-12
21 SOX9 SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal) 432656 46 41 43 0 1 2 1 4 37 1 0.0014 6.44e-15 5.05e-12
22 OR6C76 olfactory receptor, family 6, subfamily C, member 76 451906 61 60 5 0 0 1 2 1 57 0 0.2 6.77e-15 5.05e-12
23 TFAM transcription factor A, mitochondrial 362713 30 29 4 1 1 0 0 2 27 0 0.8 6.77e-15 5.05e-12
24 ESRRA estrogen-related receptor alpha 404129 35 34 6 3 1 1 2 3 28 0 0.72 6.88e-15 5.05e-12
25 TNFAIP6 tumor necrosis factor, alpha-induced protein 6 414475 59 59 7 2 3 0 0 3 53 0 0.65 7.55e-15 5.05e-12
26 ACVR2A activin A receptor, type IIA 769875 66 60 22 3 4 2 1 4 52 3 0.27 7.77e-15 5.05e-12
27 LCE4A late cornified envelope 4A 137108 18 18 4 2 0 1 0 1 16 0 0.93 7.88e-15 5.05e-12
28 KRTAP5-2 keratin associated protein 5-2 228158 28 28 3 0 0 0 0 1 27 0 0.7 7.99e-15 5.05e-12
29 KRTAP5-1 keratin associated protein 5-1 329652 34 34 8 0 0 3 1 0 30 0 0.23 8.88e-15 5.41e-12
30 RNF145 ring finger protein 145 981537 57 49 22 3 4 13 4 3 31 2 0.017 1.03e-14 6.08e-12
31 RPL22 ribosomal protein L22 166910 25 25 5 1 1 1 0 0 23 0 0.84 1.09e-14 6.20e-12
32 MUC2 mucin 2, oligomeric mucus/gel-forming 1580696 82 72 34 14 7 5 6 9 54 1 0.7 1.23e-14 6.81e-12
33 FAM123B family with sequence similarity 123B 1508599 65 60 49 7 5 10 3 6 41 0 0.038 2.22e-14 1.19e-11
34 TMBIM4 transmembrane BAX inhibitor motif containing 4 330486 24 24 6 1 2 1 1 1 19 0 0.34 3.10e-14 1.61e-11
35 HLA-B major histocompatibility complex, class I, B 308446 22 21 14 1 0 0 0 3 12 7 0.46 3.45e-14 1.74e-11
COSMIC analyses

In this analysis, COSMIC is used as a filter to increase power by restricting the territory of each gene. Cosmic version: v48.

Table 4.  Get Full Table Significantly mutated genes (COSMIC territory only). To access the database please go to: COSMIC. Number of significant genes found: 1678. Number of genes displayed: 10

rank gene description n cos n_cos N_cos cos_ev p q
1 PIK3CA phosphoinositide-3-kinase, catalytic, alpha polypeptide 344 220 296 107360 48747 0 0
2 APC adenomatous polyposis coli 685 839 582 409432 12807 0 0
3 ATM ataxia telangiectasia mutated 237 245 157 119560 307 0 0
4 TP53 tumor protein p53 437 824 437 402112 123325 0 0
5 PTCH1 patched homolog 1 (Drosophila) 143 256 118 124928 227 0 0
6 RB1 retinoblastoma 1 (including osteosarcoma) 139 267 122 130296 306 0 0
7 KIT v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog 161 240 105 117120 6849 0 0
8 PTEN phosphatase and tensin homolog (mutated in multiple advanced cancers 1) 333 767 323 374296 4779 0 0
9 NF1 neurofibromin 1 (neurofibromatosis, von Recklinghausen disease, Watson disease) 138 285 114 139080 200 0 0
10 EGFR epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) 115 293 93 142984 1342 0 0

Note:

n - number of (nonsilent) mutations in this gene across the individual set.

cos = number of unique mutated sites in this gene in COSMIC

n_cos = overlap between n and cos.

N_cos = number of individuals times cos.

cos_ev = total evidence: number of reports in COSMIC for mutations seen in this gene.

p = p-value for seeing the observed amount of overlap in this gene)

q = q-value, False Discovery Rate (Benjamini-Hochberg procedure)

Geneset Analyses

Table 5.  Get Full Table A Ranked List of Significantly Mutated Genesets. (Source: MSigDB GSEA Cannonical Pathway Set).Number of significant genesets found: 3. Number of genesets displayed: 10

rank geneset description genes N_genes mut_tally N n npat nsite nsil n1 n2 n3 n4 n5 n6 p_ns_s p q
1 SA_G1_AND_S_PHASES Cdk2, 4, and 6 bind cyclin D in G1, while cdk2/cyclin E promotes the G1/S transition. ARF1, ARF3, CCND1, CDK2, CDK4, CDKN1A, CDKN1B, CDKN2A, CFL1, E2F1, E2F2, MDM2, NXT1, PRB1, TP53 15 ARF1(1), CCND1(2), CDK2(5), CDK4(4), CDKN1A(2), CDKN1B(6), CDKN2A(9), CFL1(2), E2F2(7), MDM2(11), NXT1(5), PRB1(5), TP53(435) 5266161 494 339 277 99 140 106 56 85 103 4 1.1e-08 <1.00e-15 <6.16e-13
2 RNAPATHWAY dsRNA-activated protein kinase phosphorylates elF2a, which generally inhibits translation, and activates NF-kB to provoke inflammation. CHUK, DNAJC3, EIF2S1, EIF2S2, MAP3K14, NFKB1, NFKBIA, PRKR, RELA, TP53 9 CHUK(27), DNAJC3(4), EIF2S1(3), EIF2S2(2), MAP3K14(6), NFKB1(11), NFKBIA(1), RELA(10), TP53(435) 6724417 499 337 280 92 151 110 54 83 97 4 6.1e-10 3.55e-15 1.09e-12
3 TERTPATHWAY hTERC, the RNA subunit of telomerase, and hTERT, the catalytic protein subunit, are required for telomerase activity and are overexpressed in many cancers. HDAC1, MAX, MYC, SP1, SP3, TP53, WT1, ZNF42 7 HDAC1(5), MAX(5), MYC(5), SP1(7), SP3(13), TP53(435), WT1(32) 4783475 502 339 279 108 144 114 63 83 94 4 3.5e-09 5.66e-15 1.16e-12
4 TCRMOLECULE T Cell Receptor and CD3 Complex CD3D, CD3E, CD3G, CD3Z, TRA@, TRB@ 3 CD3D(5), CD3E(2), CD3G(16) 845905 23 21 8 3 1 5 2 0 15 0 0.46 0.0456 1.000
5 HSA00472_D_ARGININE_AND_D_ORNITHINE_METABOLISM Genes involved in D-arginine and D-ornithine metabolism DAO 1 DAO(11) 513817 11 11 11 1 9 0 1 1 0 0 0.11 0.152 1.000
6 HSA00785_LIPOIC_ACID_METABOLISM Genes involved in lipoic acid metabolism LIAS, LIPT1, LOC387787 2 LIAS(9), LIPT1(13) 1090040 22 19 17 1 0 3 2 6 11 0 0.13 0.217 1.000
7 APOPTOSIS_GENMAPP APAF1, BAK1, BCL2L7P1, BAX, BCL2, BCL2L1, BID, BIRC2, BIRC3, BIRC4, CASP2, CASP3, CASP6, CASP7, CASP8, CASP9, CYCS, FADD, FAS, FASLG, GZMB, IKBKG, JUN, MAP2K4, MAP3K1, MAP3K14, MAPK10, MCL1, MDM2, MYC, NFKB1, NFKBIA, PARP1, PRF1, RELA, RIPK1, TNF, TNFRSF1A, TNFRSF1B, TNFSF10, TP53, TRADD, TRAF1, TRAF2 41 APAF1(27), BAK1(2), BAX(16), BCL2(2), BCL2L1(1), BID(2), BIRC2(7), BIRC3(18), CASP2(8), CASP3(4), CASP6(3), CASP7(3), CASP8(20), CASP9(6), CYCS(1), FADD(1), FAS(9), FASLG(3), GZMB(5), IKBKG(2), JUN(2), MAP2K4(36), MAP3K1(21), MAP3K14(6), MAPK10(18), MDM2(11), MYC(5), NFKB1(11), NFKBIA(1), PARP1(17), PRF1(8), RELA(10), RIPK1(7), TNF(2), TNFRSF1A(4), TNFRSF1B(1), TNFSF10(3), TP53(435), TRAF1(7), TRAF2(8) 24988324 753 380 494 173 201 152 89 140 165 6 2.8e-08 0.620 1.000
8 INOSITOL_METABOLISM ALDH6A1, ALDOA, ALDOB, ALDOC, TPI1 5 ALDH6A1(6), ALDOA(2), ALDOB(16), ALDOC(3), TPI1(3) 2558746 30 28 26 3 6 6 2 15 1 0 0.027 0.677 1.000
9 SA_BONE_MORPHOGENETIC Bone morphogenetic protein binds to its receptor to induce ectopic bone formation and promote development of the viscera. BMP1, BMPR1A, BMPR1B, BMPR2, MADH1, MADH4, MADH6 4 BMP1(17), BMPR1A(18), BMPR1B(15), BMPR2(49) 4293349 99 71 82 16 21 16 5 21 26 10 0.039 0.689 1.000
10 HSA00031_INOSITOL_METABOLISM Genes involved in inositol metabolism ALDH6A1, TPI1 2 ALDH6A1(6), TPI1(3) 1063963 9 9 9 1 0 1 1 7 0 0 0.31 0.788 1.000

Table 6.  Get Full Table A Ranked List of Significantly Mutated Genesets (Excluding Significantly Mutated Genes). Number of significant genesets found: 0. Number of genesets displayed: 10

rank geneset description genes N_genes mut_tally N n npat nsite nsil n1 n2 n3 n4 n5 n6 p_ns_s p q
1 HSA00472_D_ARGININE_AND_D_ORNITHINE_METABOLISM Genes involved in D-arginine and D-ornithine metabolism DAO 1 DAO(11) 513817 11 11 11 1 9 0 1 1 0 0 0.11 0.15 1
2 HSA00785_LIPOIC_ACID_METABOLISM Genes involved in lipoic acid metabolism LIAS, LIPT1, LOC387787 2 LIAS(9), LIPT1(13) 1090040 22 19 17 1 0 3 2 6 11 0 0.13 0.22 1
3 INOSITOL_METABOLISM ALDH6A1, ALDOA, ALDOB, ALDOC, TPI1 5 ALDH6A1(6), ALDOA(2), ALDOB(16), ALDOC(3), TPI1(3) 2558746 30 28 26 3 6 6 2 15 1 0 0.027 0.68 1
4 HSA00031_INOSITOL_METABOLISM Genes involved in inositol metabolism ALDH6A1, TPI1 2 ALDH6A1(6), TPI1(3) 1063963 9 9 9 1 0 1 1 7 0 0 0.31 0.79 1
5 BBCELLPATHWAY Fas ligand expression by T cells induces apoptosis in Fas-expressing, inactive B cells. CD28, CD4, HLA-DRA, HLA-DRB1, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6 3 CD28(5), CD4(4), HLA-DRB1(7) 1196858 16 14 15 7 2 0 2 5 5 2 0.94 0.87 1
6 FLUMAZENILPATHWAY Flumazenil is a benzodiazepine receptor antagonist that may induce protective preconditioning in ischemic cardiomyocytes. GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6, GPX1, PRKCE, SOD1 9 GABRA1(19), GABRA2(17), GABRA3(7), GABRA4(22), GABRA5(21), GABRA6(22), GPX1(5), PRKCE(6) 5195954 119 86 107 26 31 17 14 40 17 0 0.0067 0.9 1
7 HSA00830_RETINOL_METABOLISM Genes involved in retinol metabolism ALDH1A1, ALDH1A2, BCMO1, RDH5 4 ALDH1A1(12), ALDH1A2(17), BCMO1(5), RDH5(2) 2727712 36 33 35 8 10 8 5 9 4 0 0.12 0.96 1
8 HSA00643_STYRENE_DEGRADATION Genes involved in styrene degradation FAH, GSTZ1, HGD 3 FAH(4), GSTZ1(5), HGD(6) 1586183 15 13 14 4 1 3 5 4 2 0 0.28 0.96 1
9 TCRMOLECULE T Cell Receptor and CD3 Complex CD3D, CD3E, CD3G, CD3Z, TRA@, TRB@ 2 CD3D(5), CD3E(2) 572147 7 7 4 3 0 5 1 0 1 0 0.67 0.97 1
10 HSA00430_TAURINE_AND_HYPOTAURINE_METABOLISM Genes involved in taurine and hypotaurine metabolism BAAT, CDO1, CSAD, GAD1, GAD2, GGT1, GGTL3, GGTL4 6 BAAT(3), CDO1(3), CSAD(6), GAD1(17), GAD2(15), GGT1(11) 3850754 55 48 40 11 18 6 7 11 13 0 0.079 0.98 1
Methods & Data
Methods

In brief, we tabulate the number of mutations and the number of covered bases for each gene. The counts are broken down by mutation context category: four context categories that are discovered by MutSig, and one for indel and 'null' mutations, which include indels, nonsense mutations, splice-site mutations, and non-stop (read-through) mutations. For each gene, we calculate the probability of seeing the observed constellation of mutations, i.e. the product P1 x P2 x ... x Pm, or a more extreme one, given the background mutation rates calculated across the dataset. [1]

Download Results

In addition to the links below, the full results of the analysis summarized in this report can also be downloaded programmatically using firehose_get, or interactively from either the Broad GDAC website or TCGA Data Coordination Center Portal.

References
[1] TCGA, Integrated genomic analyses of ovarian carcinoma, Nature 474:609 - 615 (2011)
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