New approach paves the way for large-scale genome integrity studies
NIEHS-funded researchers developed a high-throughput approach, called single-molecule mutation sequencing (SMM-seq), to characterize point mutations in normal cells. Point mutations occur when a single building block of DNA and its complement are added, deleted, or changed during replication. Linked to a variety of diseases, including cancer, point mutations have been difficult to study because they can be unique for each cell and occur at low frequencies.
SMM-seq includes a two-step library preparation protocol. First, an amplification process creates long single-stranded DNA molecules that contain multiple copies of each DNA fragment strung together. These copies are independent replicas of the original DNA fragment, reducing potential for errors to spread. Then, the long single-stranded DNA are individually amplified and converted into a sequencing library.
During this step, the team introduced unique molecular identifiers to each end of the DNA. These identifiers allowed the team to recognize matches to the original DNA fragment, filter out inherited mutations, and identify new mutations when comparing results against a single nucleotide polymorphisms database. They carried out proof-of-principle tests to detect both age-associated mutations and those following low-dose exposure to a compound known to cause mutations.
According to the authors, SMM-seq can detect both induced and naturally acquired point mutations in normal cells and tissues with high accuracy while being significantly more cost-effective than traditional methods. Paired with their structural variant search assay, this method is well suited to comprehensively assess genome integrity in large-scale human studies, according to the researchers.
Citation: Maslov AY, Makhortov S, Sun S, Heid J, Dong X, Lee M, Vijg J. 2022. Single-molecule, quantitative detection of low-abundance somatic mutations by high-throughput sequencing. Sci Adv 8(14):eabm3259.