Journal
DNA RESEARCH
Volume 23, Issue 6, Pages 547-559Publisher
OXFORD UNIV PRESS
DOI: 10.1093/dnares/dsw038
Keywords
artifactual mutations; DNA lesions; sequencing errors; PCR jackpot; ultrasensitive detection
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Funding
- 'Austrian Science Fund' (FWF) [P25525-B13]
- Austrian Academy of Sciences [23722]
- Austrian Marshall Plan Foundation [53772424132014]
- NIH [R01 GM116044]
- [FWF/P25525-B13]
- Austrian Science Fund (FWF) [P 23811, P 25525] Funding Source: researchfish
- Austrian Science Fund (FWF) [P25525] Funding Source: Austrian Science Fund (FWF)
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The need in cancer research or evolutionary biology to detect rare mutations or variants present at very low frequencies (<10(-5)) poses an increasing demand on lowering the detection limits of available methods. Here we demonstrated that amplifiable DNA lesions introduce important error sources in ultrasensitive technologies such as single molecule PCR (smPCR) applications (e.g. droplet-digital PCR), or next-generation sequencing (NGS) based methods. Using templates with known amplifiable lesions (8-oxoguanine, deaminated 5-methylcytosine, uracil, and DNA heteroduplexes), we assessed with smPCR and duplex sequencing that templates with these lesions were amplified very efficiently by proofreading polymerases (except uracil), leading to G->T, and to a lesser extent, to unreported G->C substitutions at 8-oxoguanine lesions, and C->T transitions in amplified uracil containing templates. Long heat incubations common in many DNA extraction protocols significantly increased the number of G->T substitutions. Moreover, in similar to 50-80% smPCR reactions we observed the random amplification preference of only one of both DNA strands explaining the known 'PCR jackpot effect', with the result that a lesion became indistinguishable from a true mutation or variant. Finally, we showed that artifactual mutations derived from uracil and 8-oxoguanine could be significantly reduced by DNA repair enzymes.
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