Cancer predisposition syndromes mediated by recessive cancer genes generate tumors via somatic variants (second hits) in the unaffected allele. Second hits may or may not be sufficient for neoplastic transformation. Here we performed whole-genome and whole-exome sequencing on 479 tissue biopsies from a child with neurofibromatosis type 1, a multisystem cancer-predisposing syndrome mediated by constitutive monoallelic NF1 inactivation. We identified multiple independent NF1 driver variants in histologically normal tissues, but not in 610 biopsies from two nonpredisposed children. We corroborated this finding using targeted duplex sequencing, including a further nine adults with the same syndrome. Overall, truncating NF1 mutations were under positive selection in normal tissues from individuals with neurofibromatosis type 1. We demonstrate that normal tissues in neurofibromatosis type 1 commonly harbor second hits in NF1 , the extent and pattern of which may underpin the syndrome’s cancer phenotype.

Cancer predisposition syndromes mediated by recessive cancer genes generate tumours via somatic variants (second hits) in the unaffected allele. Second hits may or may not be sufficient for neoplastic transformation. Here, we performed whole genome and exome sequencing on 479 tissue biopsies from a child with neurofibromatosis type 1, a multi-system cancer-predisposing syndrome mediated by constitutive monoallelic NF1 inactivation. We identified multiple independent NF1 driver variants in histologically normal tissues, but not in 610 biopsies from two non-predisposed children. We corroborated this finding using targeted duplex sequencing, including a further nine adults with the same syndrome. Overall, truncating NF1 mutations were under positive selection in normal tissues from individuals with neurofibromatosis type 1. We demonstrate that normal tissues in neurofibromatosis type 1 commonly harbour second hits in NF1 , the extent and pattern of which may underpin the syndrome’s cancer phenotype. ### Competing Interest Statement IM is a co-founder and consultant of Quotient Therapeutics. DH provides consultancy to AstraZeneca/MedImmune, Alexion Pharmaceuticals, Bayer, Biodexa, Roche/Genentech, and Novartis, as well as expert testimony to AstraZeneca and Novartis, and his expenses are covered by Alexion Pharmaceuticals, Boehringer Ingelheim, Roche/Genentech, and Novartis.

The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans 1 – 7 . Comparative analyses can shed light on the diversity of mutagenesis across species, and on long-standing hypotheses about the evolution of somatic mutation rates and their role in cancer and ageing. Here we performed whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found that somatic mutagenesis was dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans 8 , although the relative contribution of each signature varied across species. Notably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied showing a comparable association. Despite widely different life histories among the species we examined—including variation of around 30-fold in lifespan and around 40,000-fold in body mass—the somatic mutation burden at the end of lifespan varied only by a factor of around 3. These data unveil common mutational processes across mammals, and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.

Abstract The diversity in somatic variation across human tissues outshining human cancers has only recently been started to be appreciated. In particular, healthy individuals carry large numbers of cells and clonal expansions with mutations in driver genes commonly observed in cancer. We previously demonstrated that the majority of epithelial cells in the proximal airway accumulate DNA damage proportionally to an individual's smoking habits. However, the analysis of whole genomes indicated the abundance of a population of cells with a near-normal mutation burden, mirroring cellular populations found in never-smoking individuals. In contrast to the proximal airway, the genomics of cells in the distal airway of healthy individuals with diverse smoking histories, and thus the foundation of lung adenocarcinoma (LUAD), has not been investigated yet. To assess the influence of smoking on the distal airway and to compare the genomics of proximal and distal airway in the lung, we established an experimental procedure to create single-cell derived alveolar type II (AT2) organoids from primary human tissue of 9 patients. In particular, we focus on AT2 cells, given their hypothesised role as a potential cell of origin for LUAD. Whole genome sequencing (WGS) of >400 organoids was utilised to assess the landscape of somatic variation, including single nucleotide variants (SNVs), insertions and deletions (indels), copy number and structural variation (CNVs, SVs). Focusing on SNVs, we found that the mutation burden of never-smokers is significantly lower compared to ex- and current-smokers. Emphasising the effect of tobacco smoking on the distal airway, this difference was driven by the presence of mutations related to SBS4 and SBS92, mutational signatures associated with tobacco smoking. Interestingly, and in contrast to the proximal airway, we did not find a difference in the mutation burden of ex- and current-smokers, suggesting that smoking damage to alveolar cells is more severe and long-term. Lastly, to characterise the driver mutation landscape in depth, we used targeted single-molecule sequencing of 250 known cancer genes to profile 15 AT2 single-cell suspensions as well as 22 frozen parenchyma biopsies. To date, our preliminary results indicate positive selection on lineage defining mutations including SFTPB and SFTPC, coding for surfactant as well as TP53. As we begin to explore these data further, we expect this work to provide unprecedented insights into the contribution of somatic and driver mutations to the earliest stages of lung cancer development. Citation Format: Moritz Jakob Przybilla, Amany Ammar, Ryan Chuen Khaw, Andrew R. Lawson, Pantelis Nicola, Kate Davies, Zoe Frazer, Kate H. Gowers, Timothy M. Butler, Sarah E. Clarke, Inigo Martincorena, Sam M. Janes, Peter J. Campbell. Somatic mutations in single-cell derived alveolar organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 218.

Tumour behaviour is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To better understand these dependencies within the wider micro-environment, we studied over 270,000 single cell transcriptomes and 100 micro-dissected whole exomes from 12 patients with kidney tumours, prior to validation through the use of spatial transcriptomics. Tissues were sampled from multiple regions of the tumour core, the tumour-normal interface, normal surrounding tissues, and peripheral blood. We found the tissue-type location of CD8+ T cell clonotypes largely defined their exhaustion state, with intra-tumoural spatial heterogeneity that is not well-explained by somatic heterogeneity. De novo mutation calling from single cell RNA sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We discovered six conserved meta-programmes that distinguish tumour cell function. An epithelial-mesenchymal transition meta-programme, highly enriched at the tumour-normal interface appears co-localised with IL1B expressing macrophages, which could represent a plausible therapeutic target.Funding Information: This work was supported by Cancer Research UK/Royal College of Surgeons Clinician Scientist Fellowship (T.J.M: C63474/A27176), British Heart Foundation (R.L), the National Institute of Health Research (NIHR) Cambridge Biomedical Research Centre and the NIHR Blood and Transplant Research Unit (J.R.F. and M.R.C), Kidney Research UK Clinical PhD Fellowship (K.W.L.: TF_013_20171124), Wellcome Science Strategic Award for the Human Cell Atlas (G.S.B), Medical Research Council Human Cell Atlas Research Grant (M.R.C.: MR/S035842/1), Cancer Research UK Cambridge Centre (A.W.: C9685/A25177), Kidney Cancer UK and Facingup2Kidney cancer (M.G.B.T). The Wellcome Sanger Institute is supported by core funding from the Wellcome Trust (206194).Conflict of Interests:In the past 3 years, S.A.T has consulted for Roche and Genentech and is a Scientific Advisory Board member of Qiagen, Foresite labs, Biogen and GSK, as well as a consultant and equity holder as co-founder of Transition Bio. All other authors declare no competing interests.Ethical Approval: Human kidney and tumour tissues were collected through studies approved by UK NHS research ethics committees. All adult kidneys samples, except PD44967 were collected from patients enrolled in the DIAMOND study; Evaluation of biomarkers in urological disease (NHS National Research Ethics Service reference 03/018). Tumour PD44967 was collected from a patient enrolled in Characterisation of the immunological and biological markers of Renal cancer progression (NHS National Research Ethics Service reference 16/WS/0039).

Germ cell tumours (GCTs) are a collection of benign and malignant neoplasms derived from primordial germ cells. They are uniquely able to recapitulate embryonic and extraembryonic tissues, which carries prognostic and therapeutic significance. The developmental pathways underpinning GCT initiation and histogenesis are incompletely understood. Here, we study the relationship of histogenesis and clonal diversification in GCTs by analysing the genomes and transcriptomes of 547 microdissected histological units. We find no correlation between genomic and histological heterogeneity. However, we identify unifying features including the retention of fetal developmental transcripts across tissues, expression changes on chromosome 12p, and a conserved somatic evolutionary sequence of whole genome duplication followed by clonal diversification. While this pattern is preserved across all GCTs, the developmental timing of the duplication varies between prepubertal and postpubertal cases. In addition, tumours of younger children exhibit distinct substitution signatures which may lend themselves as potential biomarkers for risk stratification. Our findings portray the extensive diversification of GCT tissues and genetic subclones as randomly distributed, while identifying overarching transcriptional and genomic features.

Mismatch repair (MMR) is a critical defence against mutation, but we lack quantification of its activity on different DNA lesions during human life. We performed whole-genome sequencing of normal and neoplastic tissues from individuals with constitutional MMR deficiency to establish the roles of MMR components, tissue type and disease state in somatic mutation rates. Mutational signatures varied extensively across genotypes, some coupled to leading-strand replication, some to lagging-strand replication and some independent of replication, implying that the various MMR components engage different forms of DNA damage. Loss of MSH2 or MSH6 (MutSα), but not MLH1 or PMS2 (MutLα), caused 5-methylcytosine-dependent hypermutation, indicating that MutSα is the pivotal complex for repairing spontaneous deamination of methylated cytosines in humans. Neoplastic change altered the distribution of mutational signatures, particularly accelerating replication-coupled indel signatures. Each component of MMR repairs 1-10 lesions/day per normal human cell, and many thousands of additional events during neoplastic transformation. Highlights MMR repairs 1-10 lesions/day in every normal cell and thousands more in tumor cells MMR patterns and rates are shaped by genotype, tissue type and malignant transformation MSH2 and MSH6 are pivotal for repairing spontaneous deamination of methylated cytosine Replication indels and substitutions vary by leading versus lagging strand and genotype

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations(1). Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes(1-8). Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease(9-13) than in normal liver(13-16), which enables positive selection to shape the genomic landscape(9-13). Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1(S22W) hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes(17-19), and GPAM, which produces storage triacylglycerol from free fatty acids(20,21), also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.

Tumour behaviour is dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. These dependencies were examined through 270,000 single cell transcriptomes and 100 micro-dissected whole exomes obtained from 12 patients with kidney tumours. Tissue was sampled from multiple regions of tumour core, tumour-normal interface, normal surrounding tissues, and peripheral blood. We found the principal spatial location of CD8+ T cell clonotypes largely defined exhaustion state, with clonotypic heterogeneity not explained by somatic intra-tumoural heterogeneity. De novo mutation calling from single cell RNA sequencing data allows us to lineage-trace and infer clonality of cells. We discovered six meta-programmes that distinguish tumour cell function. An epithelial-mesenchymal transition meta-programme, enriched at the tumour-normal interface appears modulated through macrophage expressed IL1B, potentially forming a therapeutic target.

Over the course of an individual's lifetime, normal human cells accumulate mutations(1). Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage(2), and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma.

The extent and importance of functional heterogeneity and crosstalk between tumor cells is poorly understood. Here, we describe the generation of clonal populations from a patient-derived ovarian clear cell carcinoma model which forms malignant ascites and solid peritoneal tumors upon intraperitoneal transplantation in mice. The clonal populations are engineered with secreted Gaussia luciferase to monitor tumor growth dynamics and tagged with a unique DNA barcode to track their fate in multiclonal mixtures during tumor progression. Only one clone, CL31, grows robustly, generating exclusively malignant ascites. However, multiclonal mixtures form large solid peritoneal metastases, populated almost entirely by CL31, suggesting that transient cooperative interclonal interactions are sufficient to promote metastasis of CL31. CL31 uniquely harbors ERBB2 amplification, and its acquired metastatic activity in clonal mixtures is dependent on transient exposure to amphiregulin, which is exclusively secreted by non-tumorigenic clones. Amphiregulin enhances CL31 mesothelial clearance, a prerequisite for metastasis. These findings demonstrate that transient, ostensibly innocuous tumor subpopulations can promote metastases via "hit-and-run" commensal interactions.

To date, ontogeny of the human haematopoietic system during foetal development has been characterized mainly through careful microscopic observations. Here we used whole-genome sequencing (WGS) of 511 single-cell derived haematopoietic colonies from healthy human foetuses of 8 and 18 post-conception weeks (pcw) coupled with deep targeted sequencing of tissues of known embryonic origin to reconstruct a phylogenetic tree of blood development. We found that in healthy foetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 pcw. Using these mutations as barcodes, we timed the divergence of embryonic and extra-embryonic tissues during development and estimated the number of blood antecedents at different stages of embryonic development. Our analysis has shown that ectoderm originates from a smaller set of blood antecedents compared to endoderm and mesoderm. Finally, our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

We conducted a global characterization of the microbial communities of shipping ports to serve as a novel system to investigate microbial biogeography. The community structures of port microbes from marine and freshwater habitats house relatively similar phyla, despite spanning large spatial scales. As part of this project, we collected 1,218 surface water samples from 604 locations across eight countries and three continents to catalogue a total of 20 shipping ports distributed across the East and West Coast of the United States, Europe, and Asia to represent the largest study of port-associated microbial communities to date. Here, we demonstrated the utility of machine learning to leverage this robust system to characterize microbial biogeography by identifying trends in biodiversity across broad spatial scales. We found that for geographic locations sharing similar environmental conditions, subpopulations from the dominant phyla of these habitats (Actinobacteria, Bacteroidetes, Cyanobacteria, and Proteobacteria) can be used to differentiate 20 geographic locations distributed globally. These results suggest that despite the overwhelming diversity within microbial communities, members of the most abundant and ubiquitous microbial groups in the system can be used to differentiate a geospatial location across global spatial scales. Our study provides insight into how microbes are dispersed spatially and robust methods whereby we can interrogate microbial biogeography. IMPORTANCE Microbes are ubiquitous throughout the world and are highly diverse. Characterizing the extent of variation in the microbial diversity across large geographic spatial scales is a challenge yet can reveal a lot about what biogeography can tell us about microbial populations and their behavior. Machine learning approaches have been used mostly to examine the human microbiome and, to some extent, microbial communities from the environment. Here, we display how supervised machine learning approaches can be useful to understand microbial biodiversity and biogeography using microbes from globally distributed shipping ports. Our findings indicate that the members of globally dominant phyla are important for differentiating locations, which reduces the reliance on rare taxa to probe geography. Further, this study displays how global biogeographic patterning of aquatic microbial communities (and other systems) can be assessed through populations of the highly abundant and ubiquitous taxa that dominant the system.

Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100–1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2–3 weeks would be a more typical turnaround time.

Abstract Background: Only 1-2 % of all breast cancers present as synchronous bilateral breast cancers. These tumors may be clonally related or clonally independent. Previous analyses of synchronous bilateral breast cancers were limited by the small sample size and lack of comprehensive genomic analysis. Comparative analysis of genomic alternations in these tumors presents a unique opportunity to address the following important questions: (1) are bilateral breast cancers clonally related, and (2) do bilateral breast cancers carry molecular and genomic alterations unique from those of unilateral breast cancers. Methods: We examined synchronous left and right breast tumor samples from (16) patients collected from 1988 -1998, flash frozen, and stored at -80C in the OHSU Knight Cancer Institute BioLibrary, and 10 pairs of FFPE preserved synchronous tumors from the Kaiser Permanente NW Research Bank, collected between 2004 and 2011. These samples and matching patient demographics, clinical staging, and biomarker configuration were acquired and research conducted under IRB regulatory approval. Tumor sections were cut and tumor rich areas identified and macro-dissected. Genomic DNA was isolated and whole exon-sequencing performed. For Kaiser specimens, a benign axillary lymph node was used as a matched germ line control. Clonal relationships were determined based on distribution of somatically acquired mutations and copy number variations in the bilateral breast cancer pairs. Results: Two out of 23 synchronous bilateral breast cancers (tumors excised on the same date or within a short interval of time) are clonally related based on distribution of somatically acquired mutations and copy number variation. Two out of three metachronous bilateral breast cancers (tumors excised 6, 12 and 18 months apart) are clonally related. Each tumor of a clonally related bilateral breast pair may have a different biomarker configuration. The number of unique somatic mutations in bilateral breast cancers from four clonally related pairs, i.e. present only in tumors from one side but not the contralateral side, is small and ranges from 0 to 8. The number and distribution of somatically acquired mutations in this set of bilateral breast cancers is similar to those previously seen in unilateral breast cancers. Conclusion: To our knowledge, our study represents the largest collection of synchronous bilateral breast cancers to have received comprehensive genomic analysis. Synchronous bilateral breast cancers appear to have similar breast cancer associated mutations as have been reported with unilateral breast cancers. The molecular differences between clonally related bilateral breast cancers appears to be limited suggesting that study of these differences may reveal the molecular underpinning of breast cancer metastasis. Citation Format: Shiuh-Wen Luoh, Myron Peto, Betsy Ramsey, Carol Halsey, Timothy Butler, Paul Spellman. Comprehensive genomic analysis of synchronous bilateral breast cancer tumors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-21-04.

Many aquatic environments are at risk for oil contamination and alkanes are one of the primary constituents of oil. The alkane hydroxylase (AlkB) is a common enzyme used by microorganisms to initiate the process of alkane-degradation. While many aspects of alkane bioremediation have been studied, the diversity and evolution of genes involved in hydrocarbon degradation from environmental settings is relatively understudied. The majority of work done to-date has focused on the marine environment. Here we sought to better understand the phylogenetic diversity of alkB genes across marine and freshwater settings using culture-independent methods. We hypothesized that there would be distinct phylogenetic diversity of alkB genes in freshwater relative to the marine environment. Our results confirm that alkB has distinct variants based on environment while our diversity analyses demonstrate that freshwater and marine alkB communities have unique responses to oil amendments. Our results also demonstrate that in the marine environment, depth is a key factor impacting diversity of alkB genes.

Abstract Background Under normal physiological conditions, colonic crypts accrue ~40 somatic mutations for every year of life. That somatic mutations contribute to the development of cancer is well established, but their patterns, burden and functional consequences in diseases other than cancer have not been extensively studied and our understanding of the effects of chronic inflammation on the mutational profile and clonal structure of the colon is limited. Here, we investigated how the recurrent cycles of inflammation, ulceration and regeneration seen in IBD impact the mutational and clonal structure of intestinal epithelia. Methods We isolated and whole-genome sequenced ~400 individual colonic crypts from 46 IBD patients and compared these to ~400 crypts from 41 non-IBD controls. We compared the mutation burden, mutational signature exposure, clonal structure and cancer driver mutation landscape in crypts from actively and previously inflamed regions with crypts dissected from controls. Results We estimated the base substitution rate of affected colonic epithelial cells to be doubled after IBD onset. This change was primarily driven by acceleration of mutational processes ubiquitously observed in normal colon (Figure 1), and we did not detect an IBD-specific mutational process. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimetre-scale clonal expansions, even in the absence of mutations in KRAS, TP53 and APC (Figure 2). We discovered that mutations in ARID1A, PIGR and ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, were under positive selection in colonic crypts from IBD patients (Figure 3). With the exception of ARID1A, these genes and pathways have not been previously associated with cancer risk. A previously published mouse model of ZC3H12A suggests that LoF mutations in this gene may facilitate healing of affected mucosa without promoting tumorigenesis. This could make the encoded protein an attractive drug target. The observed enrichment of mutations in PIGR and IL17 and TLR pathways suggests that somatic mutations may initiate, maintain or perpetuate IBD pathogenesis through disruption of microbe-epithelial homeostasis. Conclusion Our results provide new insights into the consequences of chronic intestinal inflammation on the mutational profile and clonal structure of colonic epithelia. We identify the mutagens driving the increase in mutation burden and mutations which are under positive selection in the context of inflammation. Our results suggest that studying somatic mutations in the colon can reveal putative drug targets and pathogenic mechanisms for IBD.

The extent of somatic mutation and clonal selection in the human bladder remains unknown. We sequenced 2097 bladder microbiopsies from 20 individuals using targeted (n = 1914 microbiopsies), whole-exome (n = 655), and whole-genome (n = 88) sequencing. We found widespread positive selection in 17 genes. Chromatin remodeling genes were frequently mutated, whereas mutations were absent in several major bladder cancer genes. There was extensive interindividual variation in selection, with different driver genes dominating the clonal landscape across individuals. Mutational signatures were heterogeneous across clones and individuals, which suggests differential exposure to mutagens in the urine. Evidence of APOBEC mutagenesis was found in 22% of the microbiopsies. Sequencing multiple microbiopsies from five patients with bladder cancer enabled comparisons with cancer-free individuals and across histological features. This study reveals a rich landscape of mutational processes and selection in normal urothelium with large heterogeneity across clones and individuals.