Direct Quantification of in vivo Mutagenesis and Carcinogenesis Using Duplex Sequencing

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The ability to accurately measure mutations is critical for basic research and identification of potential drug and chemical carcinogens. Current methods for <jats:italic>in vivo</jats:italic> 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<jats:sup>−3</jats:sup>) of NGS obscure the true abundance of somatic mutations, which can exist at frequencies ≤1×10<jats:sup>−7</jats:sup>. 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 clear early signs of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by apparent 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 early clonal evolutionary hallmarks of carcinogenesis. Duplex Sequencing can be broadly applied to chemical safety testing, basic mutational research, and related clinical uses.</jats:p><jats:sec><jats:title>SIGNIFICANCE STATEMENT</jats:title><jats:p>Error-corrected next generation sequencing (ecNGS) can be used to rapidly detect and quantify the <jats:italic>in vivo</jats:italic> 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.</jats:p></jats:sec>

Signaling amplitude molds theRasmutation tropism of urethane

“…Mutation frequencies based on MDS sequencing of Kras exon 1 and exon 2 were averaged for A→T transversions, log 10 transformed, and displayed in a heatmap format. This revealed a trend towards A→T transversions within the context of a 5’ C in both the native and ex3op alleles of Kras specifically in urethane-exposed mice ( Figure 3B ), consistent with previously identified bias for urethane (Li et al, 2020; Valentine et al, 2020; Westcott et al, 2015). Moreover, the frequency of these C A →C T mutations, which give rise to Q 61 L (CA 182 A→C T A), are significantly higher than G G →G A mutations, which give rise to G 12 D (GG35T→G A T) and G 13 D (GG38C→G A C), in both Kras nat/nat and Kras ex3op/ex3op mice ( Figure 3C ).…”

“…Mutation frequencies based on MDS sequencing of Kras exon 1 and exon 2 were averaged for AT transversions, log10 transformed, and displayed in a heatmap format. This revealed a trend towards AT transversions within the context of a 5' C in both the native and ex3op alleles of Kras specifically in urethane-exposed mice ( Figure 3B), consistent with previously identified bias for urethane (Li et al, 2020;Valentine et al, 2020;Westcott et al, 2015).…”

“…These oncogenic mutations are also a strong match to the mutation signature of this carcinogen. Urethane-induced mutations conform to the A→T/G consensus sequence derived from comprehensive whole-exome sequencing urethane-induced tumors (Westcott et al, 2015), and the even more restricted C A N→C T/G N sequence determined shortly after urethane exposure by different ultra-sensitive sequencing approaches (Li et al, 2020; Valentine et al, 2020), which at the position C A 182 A gives rise to a C T/G A mutation encoding Q 61 L/R oncogenic mutations. We thus capitalized on the extreme mutational tropism of urethane for Kras Q61L/R -mutant pulmonary tumors to elucidate the effect of a normal cell response on the selection of initiating mutations.…”

Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing

“…Final filtered and decontaminated error-corrected alignments for all 62 mouse samples in the BAM file format are deposited in the Sequence Read Archive under BioProject accession no. PRJNA673916 ( 66 ).…”

PacBio sequencing detects genome‐wide ultra‐low‐frequency substitution mutations resulting from exposure to chemical mutagens

“…Although clone-sequencing detects genome-wide mutations accurately, it is dependent on generating clones from single cells in vitro, something that is often not possible. Error-corrected NGS, on the other hand, tags DNA with adapters containing unique barcodes (Schmitt et al, 2012;Valentine 3rd et al, 2020). The resulting chimeric DNA molecules are subject to PCR amplification, enrichment, and sequencing.…”