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Direct Quantification of in Vivo Mutagenesis and Carcinogenesis Using Duplex Sequencing

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Direct Quantification of in Vivo Mutagenesis and Carcinogenesis Using Duplex Sequencing Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing Charles C. Valentine IIIa, Robert R. Youngb, Mark R. Fieldenc,1, Rohan Kulkarnib,2, Lindsey N. Williamsa, Tan Lia, Sheroy Minocherhomjic, and Jesse J. Salka,3 aTwinStrand Biosciences, Seattle, WA 98121; bMilliporeSigma/BioReliance Toxicology Testing Services, Rockville, MD 20850; and cAmgen Research, Amgen, Thousand Oaks, CA 91320 Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved November 9, 2020 (received for review July 29, 2020) The ability to accurately measure mutations is critical for basic mutagenic potential as surrogate endpoints for regulatory research and identifying potential drug and chemical carcinogens. decision-making (7, 8). The most rapid and inexpensive of such Current methods for in vivo quantification of mutagenesis are methods include in vitro bacterial-based mutagenesis assays (e.g., limited because they rely on transgenic rodent systems that are the Ames test). Other in vitro and in vivo assays for mutation, low-throughput, expensive, prolonged, and do not fully represent chromosomal aberration induction, strand breakage, and for- other species such as humans. Next-generation sequencing (NGS) mation of micronuclei are also available; however, their sensi- is a conceptually attractive alternative for detecting mutations in tivity and specificity for predicting human cancer risk is only the DNA of any organism; however, the limit of resolution for modest. −3 standard NGS is poor. Technical error rates (∼1 × 10 ) of NGS In vivo, internationally accepted (5) mutagenesis assays using obscure the true abundance of somatic mutations, which can exist transgenic rodents (TGR) provide a powerful approximation of −7 at per-nucleotide frequencies ≤1 × 10 . Using duplex sequencing, oncogenic risk, as they reflect whole-organism biology, but are an extremely accurate error-corrected NGS (ecNGS) technology, also highly complex test systems (9). TGR mutagenesis assays we were able to detect mutations induced by three carcinogens require maintenance of multiple generations of animals bearing in five tissues of two strains of mice within 31 d following expo- an artificial reporter gene, animal exposure to the test com- sure. We observed a strong correlation between mutation induc- pound, euthanasia and necropsy several weeks after exposure, tion measured by duplex sequencing and the gold-standard isolation of the integrated genetic reporter by phage packaging, GENETICS transgenic rodent mutation assay. We identified exposure- and transfection of the phage into Escherichia coli for plaque specific mutation spectra of each compound through trinucleotide counting on many Petri dishes under permissive and nonper- patterns of base substitution. We observed variation in mutation missive selection conditions to finally obtain a mutant frequency susceptibility by genomic region, as well as by DNA strand. We readout. Although effective, the infrastructure and expertise required also identified a primordial marker of carcinogenesis in a cancer- predisposed strain of mice, as evidenced by clonal expansions of for managing a protocol which carries host DNA through three cells carrying an activated oncogene, less than a month after car- kingdoms of life has hindered ubiquitous adoption. cinogen exposure. These findings demonstrate that ecNGS is a Directly measuring ultrarare somatic mutations from extracted powerful method for sensitively detecting and characterizing mu- DNA while not being restricted by genomic locus, tissue, or tagenesis and the early clonal evolutionary hallmarks of carcino- genesis. Duplex sequencing can be broadly applied to basic Significance mutational research, regulatory safety testing, and emerging clinical applications. Error-corrected next-generation sequencing (ecNGS) can be used to rapidly detect and quantify the in vivo mutagenic im- error-corrected sequencing | genotoxicity | genetic toxicology | preclinical pact of environmental exposures or endogenous processes in cancer risk assessment | DNA repair any tissue, from any species, at any genomic location. The greater speed, higher scalability, richer data outputs, and cross- arcinogenesis is rooted in somatic evolution. Cell pop- species and cross-locus applicability of ecNGS compared to Culations bearing stochastically arising genetic mutations un- existing methods make it a powerful new tool for mutational dergo iterative waves of natural selection that enrich for mutants research, regulatory safety testing, and emerging clinical which confer a phenotype of preferential survival or proliferation applications. (1). The probability of cancer can be increased by carcinogens— Author contributions: C.C.V., R.R.Y., M.R.F., R.K., S.M., and J.J.S. designed research; C.C.V., exogenous exposures that either increase the abundance of mutations L.N.W., and T.L. performed research; C.C.V. and R.R.Y. contributed new reagents/analytic or facilitate a cell’s ability to proliferate upon selective pressures. tools; C.C.V. and J.J.S. analyzed data; and C.C.V. and J.J.S. wrote the paper. Many chemicals induce DNA damage, thereby increasing the rate of Competing interest statement: C.C.V., L.N.W., T.L., and J.J.S., are employees and equity potentially oncogenic DNA replication errors (2). The same is true holders at TwinStrand Biosciences Inc. and are authors on one or more duplex sequenc- for many forms of radiation (3). Nonmutagenic and nongenotoxic ing-related patents. R.R.Y. is an employee of MilliporeSigma. At the time the study was conducted, R.K. was an employee of MilliporeSigma but is now an employee of EMD carcinogens act through a variety of secondary mechanisms such as Serono. MilliporeSigma and EMD Serono are independent business units of Merck KGaA, inhibition of the immune system, cell-cycle overdrive to bypass nor- Darmstadt, Germany. S.M. is an employee of Amgen. M.R.F. was an employee of Amgen mal DNA replication checkpoints, and induction of inflammation at the time of the study and is currently an employee of Expansion Therapeutics. which may lead to both increased cellular proliferation and DNA This article is a PNAS Direct Submission. damage, among others (4). This open access article is distributed under Creative Commons Attribution License 4.0 Preclinical genotoxicity and carcinogenicity testing of new (CC BY). compounds is often required before regulatory authority ap- 1Present address: Expansion Therapeutics, San Diego, CA 92121. proval and subsequent human exposure (5, 6). However, current 2Present address: EMD Serono Research and Development Institute, Billerica, MA 01821. testing standards are slow and expensive; even in rodents, it takes 3To whom correspondence may be addressed. Email: [email protected]. years to reach the endpoint of tumor formation. Over the past 50 This article contains supporting information online at https://www.pnas.org/lookup/suppl/ y, a variety of approaches have been developed to more quickly doi:10.1073/pnas.2013724117/-/DCSupplemental. assess biomarkers of cancer risk by assaying DNA reactivity or www.pnas.org/cgi/doi/10.1073/pnas.2013724117 PNAS Latest Articles | 1of12 Downloaded by guest on October 1, 2021 organism (i.e., could be equally applied to rodents or humans) is Mutagen appealing yet is currently impossible with conventional next- Hours generation DNA sequencing (NGS). Standard NGS has a tech- − nical error rate (∼1 × 10 3) well above the true per-nucleotide − mutant frequency of normal tissues (<1 × 10 7) (10). New technologies for error-corrected next generation sequencing Days Necropsy and (ecNGS) have shown great promise for low-frequency mutation harvest target tissues detection in fields such as oncology and, conceptually, could be applied to genetic toxicology (11, 12). Duplex sequencing (DS), in particular, is an error-correction method that achieves ex- Extract DNA tremely high accuracy by comparing reads derived from both original strands of DNA molecules to produce duplex consensus sequences that better represent the true sequence of the source DNA molecule. DS achieves a sensitivity and specificity several Weeks Isolate and purify Library prep. with orders of magnitude greater than other methods that do not plasmid fragment duplex adapters leverage paired-strand information; it is uniquely able to resolve and sequencing mutants at the real-world frequencies produced by mutagens: on Package phage, the order of 1 in 10 million (13). infect, and In this study we tested the feasibility of using DS to measure expand in E. coli the effect of genotoxicants in vivo. We assessed the DNA of two strains of mice which were treated with three different mutagenic Transgenic carcinogens, each with distinct modes of action, and examined plaque assay five different tissue types, to generate a total of almost 10 billion error-corrected nucleotides’ worth of data. In addition to com- Detailed mutant paring mutant frequencies with those obtained from a gold- frequency, spectrum & genomic context data standard TGR assay, we explored data types not possible with Months traditional assays, including mutant spectra, trinucleotide signa- Basic mutant tures, and variations in the relative mutagenicity around the frequency data genome. Our findings illustrate the richness of genotoxicity data that can be obtained directly from genomic DNA. Finally, we Years highlight a unique opportunity to apply ecNGS to drug and chemical safety testing for the concurrent detection of both 2 year Transgenic
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