Direct Quantification of in vivo Mutagenesis and Carcinogenesis Using Duplex Sequencing

Abstract

ABSTRACT

The ability to accurately measure mutations is critical for basic research and identification of potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and don’t fully represent other species such as humans. Next generation sequencing (NGS) is a conceptually attractive alternative for mutation detection in the DNA of any organism, however, the limit of resolution for standard NGS is poor. Technical error rates (~1×10⁻³) of NGS obscure the true abundance of somatic mutations, which can exist at pernucleotide frequencies ≤1×10⁻⁷. Using Duplex Sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by 3 carcinogens in 5 tissues of 2 strains of mice within 31 days following exposure. We observed a strong correlation between mutation induction measured by Duplex Sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified the primordial signs of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex Sequencing can be broadly applied to chemical safety testing, basic mutational research, and related clinical uses.

SIGNIFICANCE STATEMENT Error-corrected next generation sequencing (ecNGS) can be used to rapidly detect and quantify the in vivo mutagenic impact of environmental exposures or endogenous processes in any tissue, from any species, at any genomic location. The greater speed, higher scalability, richer data outputs, as well as cross-species and cross-locus applicability of ecNGS compared to existing methods make it a powerful new tool for mutational research, regulatory safety testing, and emerging clinical applications.

Competing Interest Statement

CCV, LNW, TL and JJS, are employees and equity holders at TwinStrand Biosciences Incorporated. RRY is an employee of MilliporeSigma. At the time the study was conducted, RK was an employee of MilliporeSigma but is now an employee of EMD Serono. MilliporeSigma and EMD Serono are independent business units of Merck KGaA, Darmstadt Germany. SM is an employee of Amgen. MRF was an employee of Amgen at the time of the study and is currently an employee of Expansion Therapeutics.

Footnotes

* Text was reduced in length to suit journal requirements but no change to scientific content/conclusion. Small polish to grammar, syntax, organization to ready it for publication.

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ABBREVIATIONS

B[α]P

Benzo[α]pyrene

DS

Duplex Sequencing

ecNGS

Error-Corrected Next Generation Sequencing

ENU

N-Ethyl-N-Nitrosourea

MF

Mutant Frequency

SNV

Single Nucleotide Variant

TCR

Transcription-Coupled Repair

TGR

Transgenic Rodent

TPM

Transcripts per million

VC

Vehicle Control

VAF

Variant Allele Frequency