Abstract
Next generation sequencing (NGS) technologies have dramatically improved studies in biology and biomedical science. However, no optimal NGS approach is available to conveniently analyze low frequency mutations caused by DNA damage treatments. Here, by developing an exquisite ultra-sensitive NGS (USNGS) platform “EasyMF” and incorporating it with a widely used supF shuttle vector-based mutagenesis system, we can conveniently dissect roles of lesion bypass polymerases in damage-induced mutagenesis. In this improved mutagenesis analysis pipeline, the initial steps are the same as in the supF mutation assay, involving damaging the pSP189 plasmid followed by its transfection into human 293T cells to allow replication to occur. Then “EasyMF” is employed to replace downstream MBM7070 bacterial transformation and other steps for analyzing damage-induced mutation frequencies and spectra. This pipeline was validated by using UV damaged plasmid after its replication in lesion bypass polymerase-deficient 293T cells. The increased throughput and reduced cost of this system will allow us to conveniently screen regulators of translesion DNA synthesis pathway and monitor environmental genotoxic substances, which can ultimately provide insight into the mechanisms of genome stability and mutagenesis.
Highlights
Genomic DNA is constantly endangered by a variety of exogenous and endogenous agents, including ultraviolet (UV) radiation, X-ray, and chemical reagents
The sampled sequencing data indicated that even 50 Mega base (Mb) data would be enough to detect the distinct effects of translesion DNA synthesis (TLS) polymerases on UV-induced mutagenesis, which means the sequencing cost would be very low
We developed an updated ultra-sensitive next generation sequencing (USNGS) approach “Easy Mutation Frequency detection platform” (EasyMF)”, through which we figured out the mutation frequency of UV (220 J/m2) damaged pSP189 plasmid in control cells was around 1.0E-04, with 99% minor allele frequency (MAF) were lower than 0.1%
Summary
The supF mutation assay is widely employed to examine the effects of lesion bypass DNA polymerases on damaged-induced mutagenesis. The sonication fragmentation method still resulted in certain degree of background error rates in pSP189 plasmid for two types of mutation (C = > G and G = > C) To validate this conclusion, we performed tens of experiments to prepare “EasyMF” libraries of the plasmid DNA extracted from 293T cells by sonication and enzyme digestion, respectively. We have established an improved experimental and data processing platform for the rapid analysis of mutations on the supF shuttle vector in mammalian cells We believe this powerful approach will allow us to conveniently screen regulators of TLS pathway and provide insight into the mechanisms of genome stability and mutagenesis. Our experimental protocol and analysis pipeline could be used in other applications of detecting ultra-low frequency mutations
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