Adopting Duplex Sequencing™ Technology for Genetic Toxicity Testing: A Proof-of-Concept Mutagenesis Experiment with...

Area of Science:

• Genomics and Molecular Biology
• Toxicology and Pharmacology
• Biotechnology

Background:

• Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method enabling highly accurate detection of somatic mutations.
• Current in vivo gene mutation assays have limitations in sensitivity and speed.
• A DuplexSeq-based mutagenesis assay was developed for genetic toxicity testing in rats.

Approach:

• A time-course study was conducted on male Hsd:Sprague Dawley SD rats exposed to N-ethyl-N-nitrosourea (ENU).
• Mutation frequency (MF) and spectrum were analyzed in stomach, bone marrow, blood, and liver tissues at various time points (3 h, 24 h, 7 d, 28 d).
• Interlaboratory analysis was performed to assess reproducibility.

Key Points:

• Significant increases in MF were detected as early as 24 h post-exposure in stomach and bone marrow.
• The canonical ENU mutational signature was established in all four tissues by 7 d.
• DuplexSeq demonstrated remarkable reproducibility for MF and spectrum across laboratories.

Conclusions:

• DuplexSeq is an ultra-accurate NGS technology for direct mutation quantification.
• Early detection of ENU-dependent mutagenesis was achieved in proliferative tissues within 24 hours.
• The Rat-50 Mutagenesis Assay using DuplexSeq shows promise for genetic toxicology applications.