DNA mismatch and damage patterns revealed by single-molecule sequencing

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Genomic mutations accumulate throughout life, leading to cancer and other diseases.
• Most mutations initiate as single-strand DNA events, but current sequencing methods struggle to resolve them.
• Understanding these initial events is crucial for deciphering mutation origins.

Purpose of the Study:

• To develop a novel sequencing technology capable of detecting single-strand DNA damage and mismatches with high fidelity.
• To characterize single-strand mutation signatures and link them to known double-strand mutational signatures.
• To investigate mutation mechanisms in various contexts, including cancer and aging.

Main Methods:

• Development of Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq), a single-molecule, long-read sequencing method.
• Profiling of 134 diverse tissue samples, including those from individuals with cancer predisposition syndromes.
• Analysis of single-strand mismatch and damage signatures, including cytosine deamination and APOBEC3A activity.

Main Results:

• HiDEF-seq achieves single-molecule fidelity for base substitutions and cytosine deamination.
• Established correspondences between single-strand and double-strand mutational signatures, resolving initiating lesions.
• Identified distinct single-strand mismatch patterns in tumors with different repair deficiencies and defined an APOBEC3A damage signature.
• Provided insights into mutagenic mechanisms in the mitochondrial genome.

Conclusions:

• HiDEF-seq enables the detection of initial single-strand DNA events at unprecedented resolution.
• This technology can elucidate the origins of mutations in cancer, aging, and other disease contexts.
• Resolving single-strand events is key to understanding the complete mutation process beyond double-strand mutations.