Extended Data Fig. 5: Whole exome sequencing, whole genome sequencing, and chromosomal alteration of surviving tumors.
From: Mutant clones in normal epithelium outcompete and eliminate emerging tumours
(a) Wild-type mice received DEN for two months and the esophagus was collected 9 or 18 months after treatment (n=49 tumors from 16 mice). Tissues were stained for Dapi (blue) and KRT6 (red) and confocal imaged to identify tumors. Scale-bars=2mm (main), 150µm (inset). Individual tumors were manually cut under a fluorescent microscope, digested and separated in triplicates. Each triplicate was whole genome amplified (WGA) and whole exome sequenced. To exclude artefactual SNVs generated during WGA, only mutations shared by all three amplified triplicates were considered for further analysis. A total of 32,736 mutations, including silent, missense, nonsense and splice mutations and indels were identified. (b) Cumulative sequencing coverage of the whole exome triplicate samples. (c) Number of synonymous and non-synonymous mutations per tumor, ranked by mutation burden. (d) Distribution of the variant allele fraction (VAF) for the mutations common within triplicates in each tumor. (e) Percentage of silent, missense, nonsense and splice mutations and indels for individual tumors and for all tumors combined. (f) Mutational spectrum of tumors. The bar plots illustrate the percentage of mutations in each of the 96 possible trinucleotides. (g) dN/dS ratios for missense and truncating (nonsense + essential splice site) substitutions indicating genes under significant positive selection in the tumors (q<0.05, calculated with R package dNdScv26). (h) Analysis of chromosomal copy number alterations (CNAs) by whole genome sequencing of 1-year post-DEN tumors (n=64 tumors from 9 mice). Only 2 tumors, MD5924e and MD5928e, exhibited CNAs. (i) Summary of chromosomal alterations found by whole exome sequencing data of 9 or 18 months post-DEN tumors (n = 49 tumors from 16 mice). Only alterations present in all 3 whole genome amplified triplicates (j) were considered valid calls. 8140nT2 (top) is a representative example of a tumor without chromosomal alterations. 2 tumors, n2T1 and n34_T1, showed small alterations.
