iMUT-seq mapping of DSB-induced mutations with high sensitivity at single-nucleotide resolution.

DNA double-strand break (DSB)-induced mutagenesis is a critical contributor to a variety of human diseases; however, the ability to quantify these mutations is limited by the low sensitivity of current methodologies. Here we describe iMUT-seq, a technique that profiles DSB-induced mutations at high sensitivity and single-nucleotide resolution around endogenous DSBs, by coupling high-depth sequencing to restriction enzyme-inducible DSBs in human cell lines. iMUT-seq uses targeted sequencing to generate high-depth DNA libraries, enabling detection of mutations at incidence rates as low as 1 in 500,000. iMUT-seq can be used to identify novel mutagenic outcomes of DSBs and characterize previously unknown mechanisms of mutagenesis. The technique involves the extraction of genomic DNA from treated cells, followed by a multiplexed polymerase chain reaction and a series of library preparation reactions to generate a DNA library ready for sequencing on Illumina next-generation sequencing platforms. Finally, this novel approach to mutation profiling requires a specific analytical pipeline with careful considerations for the uncommon per-nucleotide nature of the data. Users will need experience in raw next-generation sequencing data analysis, and experience in DNA library preparation is beneficial. Overall, iMUT-seq has the potential to provide advanced insights into DSB mutagenesis, is a powerful tool in the study of novel DNA repair components and can be adapted to other organisms and types of DNA damage.