A unified framework for evolutionary genetic and physiological theories of aging.

Why and how we age are 2 intertwined questions that have fascinated scientists for many decades. However, attempts to answer these questions remain compartmentalized, preventing a comprehensive understanding of the aging process. We argue that the current lack of knowledge about the evolution of aging mechanisms is due to a lack of clarity regarding evolutionary theories of aging that explicitly involve physiological processes: the disposable soma theory (DST) and the developmental theory of aging (DTA). In this Essay, we propose a new hierarchical model linking genes to vital rates, enabling us to critically reevaluate the DST and DTA in terms of their relationship to evolutionary genetic theories of aging (mutation accumulation (MA) and antagonistic pleiotropy (AP)). We also demonstrate how these 2 theories can be incorporated in a unified hierarchical framework. The new framework will help to generate testable hypotheses of how the hallmarks of aging are shaped by natural selection.

The Effects of De Novo Mutation on Gene Expression and the Consequences for Fitness in Chlamydomonas reinhardtii.

Mutation is the ultimate source of genetic variation, the bedrock of evolution. Yet, predicting the consequences of new mutations remains a challenge in biology. Gene expression provides a potential link between a genotype and its phenotype. But the variation in gene expression created by de novo mutation and the fitness consequences of mutational changes to expression remain relatively unexplored. Here, we investigate the effects of >2,600 de novo mutations on gene expression across the transcriptome of 28 mutation accumulation lines derived from 2 independent wild-type genotypes of the green algae Chlamydomonas reinhardtii. We observed that the amount of genetic variance in gene expression created by mutation (Vm) was similar to the variance that mutation generates in typical polygenic phenotypic traits and approximately 15-fold the variance seen in the limited species where Vm in gene expression has been estimated. Despite the clear effect of mutation on expression, we did not observe a simple additive effect of mutation on expression change, with no linear correlation between the total expression change and mutation count of individual MA lines. We therefore inferred the distribution of expression effects of new mutations to connect the number of mutations to the number of differentially expressed genes (DEGs). Our inferred DEE is highly L-shaped with 95% of mutations causing 0-1 DEG while the remaining 5% are spread over a long tail of large effect mutations that cause multiple genes to change expression. The distribution is consistent with many cis-acting mutation targets that affect the expression of only 1 gene and a large target of trans-acting targets that have the potential to affect tens or hundreds of genes. Further evidence for cis-acting mutations can be seen in the overabundance of mutations in or near differentially expressed genes. Supporting evidence for trans-acting mutations comes from a 15:1 ratio of DEGs to mutations and the clusters of DEGs in the co-expression network, indicative of shared regulatory architecture. Lastly, we show that there is a negative correlation with the extent of expression divergence from the ancestor and fitness, providing direct evidence of the deleterious effects of perturbing gene expression.

Schrödinger's yeast: the challenge of using transformation to compare fitness among Saccharomyces cerevisiae that differ in ploidy or zygosity.

How the number of genome copies modifies the effect of random mutations remains poorly known. In yeast, researchers have investigated these effects for knock-out or other large-effect mutations, but have not accounted for differences at the mating-type locus. We set out to compare fitness differences among strains that differ in ploidy and/or zygosity using a panel of spontaneously arising mutations acquired in haploid yeast from a previous study. To ensure no genetic differences, even at the mating-type locus, we embarked on a series of transformations, which first sterilized and then temporarily introduced plasmid-borne mating types. Despite these attempts to equalize the haplotypes, fitness variation introduced during transformation swamped the differences among the original mutation-accumulation lines. While colony size looked normal, we observed a bi-modality in the maximum growth rate of our transformed yeast and determined that many of the slow growing lines were respiratory deficient ("petite"). Not previously reported, we found that yeast that were TID1/RDH54 knockouts were less likely to become petite. Even for lines with the same petite status, however, we found no correlation in fitness between the two replicate transformations performed. These results pose a challenge for any study using transformation to measure the fitness effect of genetic differences among strains. By attempting to hold haplotypes constant, we introduced more mutations that overwhelmed our ability to measure fitness differences between the genetic states. In this study, we transformed over one hundred different lines of yeast, using two independent transformations, and found that this common laboratory procedure can cause large changes to the microbe studied. Our study provides a cautionary tale of the need to use multiple transformants in fitness assays.

Contribution of Spontaneous Mutations to Quantitative and Molecular Variation at the Highly Repetitive rDNA Locus in Yeast.

The ribosomal DNA array in Saccharomyces cerevisiae consists of many tandem repeats whose copy number is believed to be functionally important but highly labile. Regulatory mechanisms have evolved to maintain copy number by directed mutation, but how spontaneous variation at this locus is generated and selected has not been well characterized. We applied a mutation accumulation approach to quantify the impacts of mutation and selection on this unique genomic feature across hundreds of mutant strains. We find that mutational variance for this trait is relatively high, and that unselected mutations elsewhere in the genome can disrupt copy number maintenance. In consequence, copy number generally declines gradually, consistent with a previously proposed model of rDNA maintenance where a downward mutational bias is normally compensated by mechanisms that increase copy number when it is low. This pattern holds across ploidy levels and strains in the standard lab environment but differs under some stressful conditions. We identify several alleles, gene categories, and genomic features that likely affect copy number, including aneuploidy for chromosome XII. Copy number change is associated with reduced growth in diploids, consistent with stabilizing selection. Levels of standing variation in copy number are well predicted by a balance between mutation and stabilizing selection, suggesting this trait is not subject to strong diversifying selection in the wild. The rate and spectrum of point mutations within the rDNA locus itself are distinct from the rest of the genome and predictive of polymorphism locations. Our findings help differentiate the roles of mutation and selection and indicate that spontaneous mutation patterns shape several aspects of ribosomal DNA evolution.

Life history and deleterious mutation rate coevolution.

The cost of germline maintenance gives rise to a trade-off between lowering the deleterious mutation rate and investing in life history functions. Therefore, life history and the mutation rate coevolve, but this coevolution is not well understood. We develop a mathematical model to analyse the evolution of resource allocation traits, which simultaneously affect life history and the deleterious mutation rate. First, we show that the invasion fitness of such resource allocation traits can be approximated by the basic reproductive number of the least-loaded class; the expected lifetime production of offspring without deleterious mutations born to individuals without deleterious mutations. Second, we apply the model to investigate (i) the coevolution of reproductive effort and germline maintenance and (ii) the coevolution of age-at-maturity and germline maintenance. This analysis provides two resource allocation predictions when exposure to environmental mutagens is higher. First, selection favours higher allocation to germline maintenance, even if it comes at the expense of life history functions, and leads to a shift in allocation towards reproduction rather than survival. Second, life histories tend to be faster, characterised by individuals with shorter lifespans and smaller body sizes at maturity. Our results suggest that mutation accumulation via the cost of germline maintenance can be a major force shaping life-history traits.

Epidermal mutation accumulation in photodamaged skin is associated with skin cancer burden and can be targeted through ablative therapy.

The main carcinogen for keratinocyte skin cancers (KCs) such as basal and squamous cell carcinomas is ultraviolet (UV) radiation. There is growing evidence that accumulation of mutations and clonal expansion play a key role in KC development. The relationship between UV exposure, epidermal mutation load, and KCs remains unclear. Here, we examined the mutation load in both murine (n = 23) and human (n = 37) epidermal samples. Epidermal mutations accumulated in a UV dose-dependent manner, and this mutation load correlated with the KC burden. Epidermal ablation (either mechanical or laser induced), followed by spontaneous healing from underlying epithelial adnexae reduced the mutation load markedly in both mouse (n = 8) and human (n = 6) clinical trials. In a model of UV-induced basal cell carcinoma, epidermal ablation reduced incident lesions by >80% (n = 5). Overall, our findings suggest that mutation burden is strongly associated with KC burden and represents a target to prevent subsequent KCs.

On aging and age-specific effects of spontaneous mutations.

Evolutionary theory assumes that mutations that cause aging either have beneficial early-life effects that gradually become deleterious with advancing age (antagonistic pleiotropy [AP]) or that they only have deleterious effects at old age (mutation accumulation [MA]). Mechanistically, aging is predicted to result from damage accumulating in the soma. While this scenario is compatible with AP, it is not immediately obvious how damage would accumulate under MA. In a modified version of the MA theory, it has been suggested that mutations with weakly deleterious effects at young age can also contribute to aging, if they generate damage that gradually accumulates with age. Mutations with increasing deleterious effects have recently gained support from theoretical work and studies of large-effect mutations. Here we address if spontaneous mutations also have negative effects that increase with age. We accumulate mutations with early-life effects in Drosophila melanogaster across 27 generations and compare their relative effects on fecundity early and late in life. Our mutation accumulation lines on average have substantially lower early-life fecundity compared to controls. These effects were further maintained throughout life, but they did not increase with age. Our results suggest that most spontaneous mutations do not contribute to damage accumulation and aging.

Modelling somatic mutation accumulation and expansion in a long-lived tree with hierarchical modular architecture.

In a long-lived organism with a modular architecture, such as trees, somatic mutations accumulate throughout the long lifespan and result in genetic mosaicism in each module within the same individual. In recent years, next-generation sequencing technology has provided a snapshot of such intra-organismal genetic variability. However, the dynamic processes underlying the accumulation and expansion of somatic mutations during the growth remain poorly understood. In this study, we constructed a model to describe these processes in a form that can be applied to a real tree. Given that the proliferation dynamics of meristematic cells vary across plant species, multiple possible processes for elongation and branching were comprehensively expressed in our model. Using published data from a poplar tree, we compared the prediction of the models with the observation and explained the cell lineage dynamics underlying somatic mutations accumulation that were not evident from the snapshot of the sequenced data. We showed that the somatic genetic drift during growth increases inter-meristem mosaicism, resulting in genetically distinct branches and less integrity within an individual tree. We also showed that the somatic genetic drift during branching leads to the mutation accumulation pattern that does not reflect the tree topology. Our modelling framework can help interpret and provide further insights into the empirical findings of genetic mosaicism in long-lived trees.

Rates and spectra of de novo structural mutations in Chlamydomonas reinhardtii.

Genetic variation originates from several types of spontaneous mutation, including single-nucleotide substitutions, short insertions and deletions (indels), and larger structural changes. Structural mutations (SMs) drive genome evolution and are thought to play major roles in evolutionary adaptation, speciation, and genetic disease, including cancers. Sequencing of mutation accumulation (MA) lines has provided estimates of rates and spectra of single-nucleotide and indel mutations in many species, yet the rate of new SMs is largely unknown. Here, we use long-read sequencing to determine the full mutation spectrum in MA lines derived from two strains (CC-1952 and CC-2931) of the green alga Chlamydomonas reinhardtii The SM rate is highly variable between strains and between MA lines, and SMs represent a substantial proportion of all mutations in both strains (CC-1952 6%; CC-2931 12%). The SM spectra differ considerably between the two strains, with almost all inversions and translocations occurring in CC-2931 MA lines. This variation is associated with heterogeneity in the number and type of active transposable elements (TEs), which comprise major proportions of SMs in both strains (CC-1952 22%; CC-2931 38%). In CC-2931, a Crypton and a previously undescribed type of DNA element have caused 71% of chromosomal rearrangements, whereas in CC-1952, a Dualen LINE is associated with 87% of duplications. Other SMs, notably large duplications in CC-2931, are likely products of various double-strand break repair pathways. Our results show that diverse types of SMs occur at substantial rates, and support prominent roles for SMs and TEs in evolution.

Identification of natural product 3, 5-diiodotyrosine as APOBEC3B inhibitor to prevent somatic mutation accumulation and cancer progression.

BACKGROUND: The development of cancer is largely dependent on the accumulation of somatic mutations, indicating the potential to develop cancer chemoprevention agents targeting mutation drivers. However, ideal cancer chemoprevention agents that can effectively inhibit the mutation drivers have not been identified yet. METHODS: The somatic mutation signatures and expression analyses of APOBEC3B were performed in patient with pan-cancer. The computer-aided screening and skeleton-based searching were performed to identify natural products that can inhibit the activity of APOBEC3B. 4-nitroquinoline-1-oxide (4-NQO)-induced spontaneous esophageal squamous cell carcinoma (ESCC) and azoxymethane/dextran sulfate sodium (AOM/DSS)-induced spontaneous colon cancer mouse models were conducted to investigate the influences of APOBEC3B inhibitor on the prevention of somatic mutation accumulation and cancer progression. RESULTS: Here, we discovered that the cytidine deaminase APOBEC3B correlated somatic mutations were widely observed in a variety of cancers, and its overexpression indicated poor survival. SMC247 (3, 5-diiodotyrosine), as a source of kelp iodine without side effects, could strongly bind APOBEC3B (KD=65 nM) and effectively inhibit its deaminase activity (IC50=1.69 µM). Interestingly, 3, 5-diiodotyrosine could significantly reduce the clusters of mutations, prevent the precancerous lesion progression, and prolong the survival in 4-NQO-induced spontaneous ESCC and AOM/DSS-induced spontaneous colon cancer mouse models. Furthermore, 3, 5-diiodotyrosine could reduce colitis, increase the proportion and function of T lymphocytes via IL-15 in tumor microenvironment. The synergistic cancer prevention effects were observed when 3, 5-diiodotyrosine combined with PD-1/PD-L1 blockade. CONCLUSIONS: This is the first prove-of-concept study to elucidate that the natural product 3, 5-diiodotyrosine could prevent somatic mutation accumulation and cancer progression through inhibiting the enzymatic activity of APOBEC3B. In addition, 3, 5-diiodotyrosine could reduce the colitis and increase the infiltration and function of T lymphocytes via IL-15 in tumor microenvironment. 3, 5-diiodotyrosine combined with PD-1/PD-L1 blockade could elicit synergistic cancer prevention effects, indicating a novel strategy for both prevent the somatic mutation accumulation and the immune-suppressive microenvironment exacerbation.

Selection in a growing colony biases results of mutation accumulation experiments.

Mutations provide the raw material for natural selection to act. Therefore, understanding the variety and relative frequency of different type of mutations is critical to understanding the nature of genetic diversity in a population. Mutation accumulation (MA) experiments have been used in this context to estimate parameters defining mutation rates, distribution of fitness effects (DFE), and spectrum of mutations. MA experiments can be performed with different effective population sizes. In MA experiments with bacteria, a single founder is grown to a size of a colony (~ 108). It is assumed that natural selection plays a minimal role in dictating the dynamics of colony growth. In this work, we simulate colony growth via a mathematical model, and use our model to mimic an MA experiment. We demonstrate that selection ensures that, in an MA experiment, fraction of all mutations that are beneficial is over-represented by a factor of almost two, and that the distribution of fitness effects of beneficial and deleterious mutations are inaccurately captured in an MA experiment. Given this, the estimate of mutation rates from MA experiments is non-trivial. We then perform an MA experiment with 160 lines of E. coli, and show that due to the effect of selection in a growing colony, the size and sector of a colony from which the experiment is propagated impacts the results. Overall, we demonstrate that the results of MA experiments need to be revisited taking into account the action of selection in a growing colony.

Identification of Warning Transition Points from Hepatitis B to Hepatocellular Carcinoma Based on Mutation Accumulation for the Early Diagnosis and Potential Drug Treatment of HBV-HCC.

The accumulation of multiple genetic mutations is essential during the occurrence and development of hepatocellular carcinoma induced by hepatitis B (HBV-HCC), but understanding their cooperative effects and identifying the warning transition point from hepatitis B to HCC are challenges. In the genomic analysis of somatic mutations of the patient with HBV-HCC in a patient-specific protein-protein interaction (ps-PPI) network, we find mutation influence can propagate along the ps-PPI network. Therefore, in the article, we got the mutation cluster as a new research unit using the Random Walks with Restarts algorithm that is used to describe the efficient boundary of mutation influences. The connection of mutation cluster leads to dysregulation of signaling pathways corresponding to HCC, while dysregulated signaling pathways accumulate gradually and experience a process from quantitative to qualitative changes including a critical mutation cluster called transition point (TP) from hepatitis B to HCC. Moreover, two subtypes of HCC patients with different prognosis and their corresponding biological and clinical characteristics were identified according to TP. The poor prognosis HCC subtype was associated with significant metabolic pathway dysregulation and lower immune cell infiltration, while we also identified several preventive drugs to block the transformation of hepatitis B to hepatocellular carcinoma. The network-level study integrated multiomics data not only showed the sequence of multiple somatic mutations and their cooperative effect but also identified the warning transition point in HCC tumorigenesis for each patient. Our study provides new insight into exploring the cooperative molecular mechanism of chronic inflammatory malignancy in the liver and lays the foundation for the development of new approaches for early prediction and diagnosis of hepatocellular carcinoma and personalized targeted therapy.

Resistance of mitochondrial DNA to cadmium and Aflatoxin B1 damage-induced germline mutation accumulation in C. elegans.

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.

Heterogeneity in viral populations increases the rate of deleterious mutation accumulation.

RNA viruses have high mutation rates, with the majority of mutations being deleterious. We examine patterns of deleterious mutation accumulation over multiple rounds of viral replication, with a focus on how cellular coinfection and heterogeneity in viral output affect these patterns. Specifically, using agent-based intercellular simulations we find, in agreement with previous studies, that coinfection of cells by viruses relaxes the strength of purifying selection and thereby increases the rate of deleterious mutation accumulation. We further find that cellular heterogeneity in viral output exacerbates the rate of deleterious mutation accumulation, regardless of whether this heterogeneity in viral output is stochastic or is due to variation in the cellular multiplicity of infection. These results highlight the need to consider the unique life histories of viruses and their population structure to better understand observed patterns of viral evolution.

Repeat sequences limit the effectiveness of lateral gene transfer and favored the evolution of meiotic sex in early eukaryotes.

The transition from prokaryotic lateral gene transfer to eukaryotic meiotic sex is poorly understood. Phylogenetic evidence suggests that it was tightly linked to eukaryogenesis, which involved an unprecedented rise in both genome size and the density of genetic repeats. Expansion of genome size raised the severity of Muller's ratchet, while limiting the effectiveness of lateral gene transfer (LGT) at purging deleterious mutations. In principle, an increase in recombination length combined with higher rates of LGT could solve this problem. Here, we show using a computational model that this solution fails in the presence of genetic repeats prevalent in early eukaryotes. The model demonstrates that dispersed repeat sequences allow ectopic recombination, which leads to the loss of genetic information and curtails the capacity of LGT to prevent mutation accumulation. Increasing recombination length in the presence of repeat sequences exacerbates the problem. Mutational decay can only be resisted with homology along extended sequences of DNA. We conclude that the transition to homologous pairing along linear chromosomes was a key innovation in meiotic sex, which was instrumental in the expansion of eukaryotic genomes and morphological complexity.

Characterization of a Treponema denticola ATCC 35405 mutant strain with mutation accumulation, including a lack of phage-derived genes.

Trepoenema denticola, a spirochetal bacterium, is associated with periodontal diseases. The type strain of the bacterium, ATCC 35405, is commonly used in a basic research. Here, we report that our stock strain derived from ATCC 35405 had a mutation on the chromosome and expressed differential characteristics from the original strain. Genome sequencing analysis revealed the lack of a phage-derived region, and over 200 mutations in the mutant strain. The mutant grew to a higher density in broth culture as compared with the origin. In addition, the mutant formed a colony on the surface of the agar medium, whereas the origin could not. On contrary, the mutant showed decreased motility and adhesion to gingival epithelial cells. There were no differences in the bacterial cell length and a chymotrypsin-like protease activity between the two strains. RNA and genome sequencing analysis could not identify the genes that introduced the phenotypic differences between the strains. This mutant is potentially useful for examining the genetic background responsible for the physiological and pathogenic characteristics of T. denticola.

Mutation accumulation opposes polymorphism: supergenes and the curious case of balanced lethals.

Supergenes offer spectacular examples of long-term balancing selection in nature, but their origin and maintenance remain a mystery. Reduced recombination between arrangements, a critical aspect of many supergenes, protects adaptive multi-trait phenotypes but can lead to mutation accumulation. Mutation accumulation can stabilize the system through the emergence of associative overdominance (AOD), destabilize the system, or lead to new evolutionary outcomes. One outcome is the formation of maladaptive balanced lethal systems, where only heterozygotes remain viable and reproduce. We investigated the conditions under which these different outcomes occur, assuming a scenario of introgression after divergence. We found that AOD aided the invasion of a new supergene arrangement and the establishment of a polymorphism. However, this polymorphism was easily destabilized by further mutation accumulation, which was often asymmetric, disrupting the quasi-equilibrium state. Mechanisms that accelerated degeneration tended to amplify asymmetric mutation accumulation between the supergene arrangements and vice-versa. As the evolution of balanced lethal systems requires symmetric degeneration of both arrangements, this leaves only restricted conditions for their evolution, namely small population sizes and low rates of gene conversion. The dichotomy between the persistence of polymorphism and degeneration of supergene arrangements likely underlies the rarity of balanced lethal systems in nature. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.

Causes of variability in estimates of mutational variance from mutation accumulation experiments.

Characteristics of the new phenotypic variation introduced via mutation have broad implications in evolutionary and medical genetics. Standardized estimates of this mutational variance, VM, span 2 orders of magnitude, but the causes of this remain poorly resolved. We investigated estimate heterogeneity using 2 approaches. First, meta-analyses of ∼150 estimates of standardized VM from 37 mutation accumulation studies did not support a difference among taxa (which differ in mutation rate) but provided equivocal support for differences among trait types (life history vs morphology, predicted to differ in mutation rate). Notably, several experimental factors were confounded with taxon and trait, and further empirical data are required to resolve their influences. Second, we analyzed morphological data from an experiment in Drosophila serrata to determine the potential for unintentional heterogeneity among environments in which phenotypes were measured (i.e. among laboratories or time points) or transient segregation of mutations within mutation accumulation lines to affect standardized VM. Approximating the size of an average mutation accumulation experiment, variability among repeated estimates of (accumulated) mutational variance was comparable to variation among published estimates of standardized VM. This heterogeneity was (partially) attributable to unintended environmental variation or within line segregation of mutations only for wing size, not wing shape traits. We conclude that sampling error contributed substantial variation within this experiment, and infer that it will also contribute substantially to differences among published estimates. We suggest a logistically permissive approach to improve the precision of estimates, and consequently our understanding of the dynamics of mutational variance of quantitative traits.

Trade-off between reducing mutational accumulation and increasing commitment to differentiation determines tissue organization.

Species-specific differences control cancer risk across orders of magnitude variation in body size and lifespan, e.g., by varying the copy numbers of tumor suppressor genes. It is unclear, however, how different tissues within an organism can control somatic evolution despite being subject to markedly different constraints, but sharing the same genome. Hierarchical differentiation, characteristic of self-renewing tissues, can restrain somatic evolution both by limiting divisional load, thereby reducing mutation accumulation, and by increasing cells' commitment to differentiation, which can "wash out" mutants. Here, we explore the organization of hierarchical tissues that have evolved to limit their lifetime incidence of cancer. Estimating the likelihood of cancer in the presence of mutations that enhance self-proliferation, we demonstrate that a trade-off exists between mutation accumulation and the strength of washing out. Our results explain differences in the organization of widely different hierarchical tissues, such as colon and blood.

MutationalPatterns: the one stop shop for the analysis of mutational processes.

BACKGROUND: The collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data. RESULTS: Here, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights. CONCLUSIONS: This novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns .