Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing

Area of Science:

• Genomics
• Toxicology
• Cancer Research

Background:

• Accurate mutation measurement is vital for research and carcinogen identification.
• Existing in vivo mutagenesis assays (transgenic rodent systems) are limited by low throughput, high cost, and poor human relevance.
• Standard next-generation sequencing (NGS) has insufficient resolution for detecting low-frequency somatic mutations due to high technical error rates.

Purpose of the Study:

• To evaluate the utility of duplex sequencing, an error-corrected NGS (ecNGS) technology, for sensitive and accurate in vivo mutation detection.
• To characterize mutation spectra and susceptibility in response to carcinogen exposure.
• To identify early molecular markers of carcinogenesis.

Main Methods:

• Utilized duplex sequencing (ecNGS) to quantify mutations induced by three carcinogens in five mouse tissues within 31 days.
• Compared duplex sequencing results with the gold-standard transgenic rodent mutation assay.
• Analyzed mutation spectra using trinucleotide patterns and assessed variations by genomic region and DNA strand.

Main Results:

• Duplex sequencing demonstrated a strong correlation with the transgenic rodent assay for mutation induction.
• Identified distinct, exposure-specific mutation spectra for each carcinogen.
• Observed variations in mutation susceptibility across genomic regions and DNA strands.
• Detected early clonal expansions of oncogene-carrying cells in a cancer-predisposed mouse strain.

Conclusions:

• Error-corrected NGS (ecNGS) via duplex sequencing is a highly sensitive and accurate method for detecting and characterizing mutagenesis in vivo.
• This technology can reveal early clonal evolutionary hallmarks of carcinogenesis.
• Duplex sequencing has broad applications in basic research, regulatory safety testing, and clinical settings.