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. 2016 Apr 28:6:25310.
doi: 10.1038/srep25310.

Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis

Kaile Wang  1   2 , Xiaolu Ma  1   2 , Xue Zhang  1   2 , Dafei Wu  1 , Chenyi Sun  1   2 , Yazhou Sun  1   2 , Xuemei Lu  1 , Chung-I Wu  1   3   4 , Caixia Guo  1 , Jue Ruan  1   5
Affiliations

Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis

Kaile Wang et al. Sci Rep. .

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.

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Figures

Figure 1
Figure 1. DNA damage-induced mutagenesis and EasyMF sequencing library preparation.
(a) Schematic diagram of mutation frequency analysis in pSP189 using traditional supF shuttle vector-based mutagenesis assay (TSMA) and “EasyMF”. The undamaged or damaged (220 J/m2 UVC) pSP189 plasmid was transfected into the indicated siRNA-treated 293T cells. Mutation frequency in pSP189 was determined after it was recovered from 293T cells through both traditional supF shuttle vector-based mutagenesis assay (TSMA) and “EasyMF”. (b) The diagram of “EasyMF”. DNA was sheared into fragments shorter than half the length of the sequencing read and circularized, then used for RCA. Next, the amplified DNA were taken to prepare standard NGS libraries. The real mutation will appear in each tandem repeats (red dots), conversely, errors resulted from the RCA or NGS library preparation only happen in some of repeats (blue dots) randomly. Since the size of sheared DNA fragments shorter than the length of a single PE read, the original DNA will be sequenced at least twice in a pair of PE reads independently to eliminate PCR and sequencing errors. The consensus sequence (CS) can be determined by align read 1 and read 2 in one pair of PE reads, and the site of CS will be given with high quality scores if it is supported by at least twice, and it will be given with the lowest quality scores if it is supported by only once.
Figure 2
Figure 2. Mutation frequency pattern in DNA damage-induced mutagenesis measured by “EasyMF”.
(a) Background mutation frequency and mutation spectrum of “EasyMF” in undamaged pSP189 from control cells. (b) Mutation frequencies in undamaged and UV damaged pSP189 plasmids were determined after they were recovered from siPolη-treated 293T cells through “EasyMF”, “EasyMF95” (mutation frequency of every base in 95bp target region), TSMA-Sbase (mutation frequency of single base measured by TSMA). siPolη and siPolη-UV represent undamaged or UV damaged plasmid transfected into siPolη-treated 293T cells, respectively, *0.01 = <p < 0.05, **p < 0.01. (c) Distribution of mutations’ MAFs of undamaged and UV damaged pSP189 plasmids in siPolη-treated 293T cells, y axis represents the proportion of mutations, and x axis represents interval of MAFs. (d) C = >T mutation frequencies at monodipyrimidine, dipyrimidine and polypyrimidine sites of undamaged plasmid in control 293T cells. (e) C = >T mutation frequencies at monodipyrimidine, dipyrimidine and polypyrimidine sites of UV damaged plasmid in control 293T cells.
Figure 3
Figure 3
Mutation frequency changes in control, Polη or REV1 knocked down cells measured by “EasyMF” (a) and TSMA (b). X axis represents different experimental replicates, while y axis represents the fold change of mutation frequency compared with corresponding siNC-UV. (c) The p-values of siPolη-UV versus siNC-UV and siREV1-UV versus siNC-UV. (d) The effects of Polη- or REV1-depletion on the base substitution frequencies of UV damaged pSP189 plasmid, one replicate was showed, another two were showed in Fig. S8. (e) The significance statistical analysis of base substitution of siPolη-UV and siREV1-UV compared with siNC-UV. Y axis is the fold changes of mutation frequency for each types of base substitutions.
Figure 4
Figure 4. Reproducibility and sensitivity of the “EasyMF”.
(a) Overview of mutation frequency in pSP189 plasmid, slide window size is 100bp. X axis is the genome position, y axis is the mutation frequency of each region. (b–d) Fold changes of mutation frequency relative to corresponding siNC-UV from sub-sampled 1 Mb, 10 Mb and 50 Mb data, 3 replicates were plotted. (e) p-values of the mutation frequency difference between undamaged and UV damaged plasmids in indicated circumstances, or siPolη-UV and siNC-UV, or siREV1-UV and siNC-UV in 1 Mb, 10 Mb and 50 Mb sub-sampled data.
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