Jesse J. Salk

Detection of ultra-rare mutations by next-generation sequencing

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ∼1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.

Mutational Heterogeneity in Human Cancers: Origin and Consequences

Cancer recapitulates Darwinian evolution. Mutations acquired during life that provide cells with a growth or survival advantage will preferentially multiply to form a tumor. As a result of The Cancer Genome Atlas Project, we have gathered detailed information on the nucleotide sequence changes in a number of human cancers. The sources of mutations in cancer are diverse, and the complexity of those found to be clonally present in tumors has increasingly made it difficult to identify key rate-limiting genes for tumor growth that could serve as potential targets for directed therapies. The impact of DNA sequencing on future cancer research and personalized therapy is likely to be profound and merits critical evaluation.

Ultra-Sensitive Sequencing Reveals an Age-Related Increase in Somatic Mitochondrial Mutations That Are Inconsistent with Oxidative Damage

Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >107 wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ∼5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G→T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G→A and T→C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.

Optimization of DNA polymerase mutation rates during bacterial evolution

Mutation rate is an important determinant of evolvability. The optimal mutation rate for different organisms during evolution has been modeled in silico and tested in vivo, predominantly through pairwise comparisons. To characterize the fitness landscape across a broad range of mutation rates, we generated a panel of 66 DNA polymerase I mutants in Escherichia coli with comparable growth properties, yet with differing DNA replication fidelities, spanning 103-fold higher and lower than that of wild type. These strains were competed for 350 generations in six replicate cultures in two different environments. A narrow range of mutation rates, 10- to 47-fold greater than that of wild type, predominated after serial passage. Mutants exhibiting higher mutation rates were not detected, nor were wild-type or antimutator strains. Winning clones exhibited shorter doubling times, greater maximum culture densities, and a growth advantages in pairwise competition relative to their precompetition ancestors, indicating the acquisition of adaptive phenotypes. To investigate the basis for mutator selection, we undertook a large series of pairwise competitions between mutator and wild-type strains under conditions where, in most cases, one strain completely overtook the culture within 18 days. Mutators were the most frequent winners but wild-type strains were also observed winning, suggesting that the competitive advantage of mutators is due to a greater probability of developing selectably advantageous mutations rather than from an initial growth advantage conferred by the polymerase variant itself. Our results indicate that under conditions where organism fitness is not yet maximized for a particular environment, competitive adaptation may be facilitated by enhanced mutagenesis.

Cancer Genome Sequencing—An Interim Analysis

With the publishing of the first complete whole genome of a human cancer and its paired normal, we have passed a key milestone in the cancer genome sequencing strategy. The generation of such data will, thanks to technical advances, soon become commonplace. As a significant number of proof-of-concept studies have been published, it is important to analyze now the likely implications of these data and how this information might frame cancer research in the near future. The diversity of genes mutated within individual tumor types, the most striking feature of all studies reported to date, challenges gene-centric models of tumorigenesis. Although cancer genome sequencing will revolutionize certain aspects of personalized care, the value of these studies in facilitating the development of new therapies, their primary goal, seems less promising. Most significantly, however, the cancer genome sequencing strategy, as currently applied, fails to characterize the most relevant genomic features of cancer-the mutational heterogeneity within individual tumors.

The influence of subclonal resistance mutations on targeted cancer therapy

Clinical oncology is being revolutionized by the increasing use of molecularly targeted therapies. This paradigm holds great promise for improving cancer treatment; however, allocating specific therapies to the patients who are most likely to derive a durable benefit continues to represent a considerable challenge. Evidence continues to emerge that cancers are characterized by extensive intratumour genetic heterogeneity, and that patients being considered for treatment with a targeted agent might, therefore, already possess resistance to the drug in a minority of cells. Indeed, multiple examples of pre-existing subclonal resistance mutations to various molecularly targeted agents have been described, which we review herein. Early detection of pre-existing or emerging drug resistance could enable more personalized use of targeted cancer therapy, as patients could be stratified to receive the therapies that are most likely to be effective. We consider how monitoring of drug resistance could be incorporated into clinical practice to optimize the use of targeted therapies in individual patients.

Decreased Mitochondrial DNA Mutagenesis in Human Colorectal Cancer

Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested. In this study, we directly measured the frequency of non-clonal (random) de novo single base substitutions in the mtDNA of human colorectal cancers. Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue. The difference in mutation burden was attributable to a reduction in C∶G to T∶A transitions, which are associated with oxidative damage. We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon. Together these findings raise the intriguing possibility that fidelity of mitochondrial genome is, in fact, increased in cancer as a result of a decrease in reactive oxygen species-mediated mtDNA damage.

Clonal expansions in ulcerative colitis identify patients with neoplasia

Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulate mutations, some of which affect growth regulation and drive successive waves of clonal evolution, whereas a far greater number are functionally neutral and serve only to passively mark expanding clones. Ulcerative colitis (UC) is an inflammatory bowel disease, in which up to 10% of patients eventually develop colon cancer. Here we have mapped mutations in hypermutable intergenic and intronic polyguanine tracts in patients with UC to delineate the extent of clonal expansions associated with carcinogenesis. We genotyped colon biopsies for length altering mutations at 28 different polyguanine markers. In eight patients without neoplasia, we detected only two mutations in a single individual from among 37 total biopsies. In contrast, for 11 UC patients with neoplasia elsewhere in the colon, we identified 63 mutations in 51 nondysplastic biopsies, and every patient possessed at least one mutant clone. A subset of clones were large and extended over many square centimeters of colon. Of these, some occurred as isolated populations in nondysplastic tissue, considerably distant from neoplastic lesions. Other large clones included regions of cancer, suggesting that the tumor arose within a preexisting clonal field. Our results demonstrate that neutral mutations in polyguanine tracts serve as a unique tool for identifying fields of clonal expansions, which may prove clinically useful for distinguishing a subset of UC patients who are at risk for developing cancer.

Two-temperature LATE-PCR endpoint genotyping

BackgroundIn conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay.ResultsTwo-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles.ConclusionSNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions.

Passenger mutations as a marker of clonal cell lineages in emerging neoplasia

Cancer arises as the result of a natural selection process among cells of the body, favoring lineages bearing somatic mutations that bestow them with a proliferative advantage. Of the thousands of mutations within a tumor, only a small fraction functionally drive its growth; the vast majority are mere passengers of minimal biological consequence. Yet the presence of any mutation, independent of its role in facilitating proliferation, tags a cells clonal descendants in a manner that allows them to be distinguished from unrelated cells. Such markers of cell lineage can be used to identify the abnormal proliferative signature of neoplastic clonal evolution, even at a stage which predates morphologically recognizable dysplasia. This article focuses on molecular techniques for assessing cellular clonality in humans with an emphasis on how they may be used for early detection of tumorigenic processes. We discuss historical as well as contemporary approaches and consider ways in which powerful new genomic technologies might be harnessed to develop a future generation of early cancer diagnostics.