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Solutions for PCR, cloning and sequencing errors in population genetic analysis

Cummings, S. M.; McMullan, M.; Joyce, D. A.; van Oosterhout, C.

Authors

S. M. Cummings

M. McMullan

C. van Oosterhout



Abstract

PCR and sequencing artefacts can seriously bias population genetic analyses, particularly of populations with low genetic variation such as endangered vertebrate populations. Here, we estimate the error rates, discuss their population genetics implications, and propose a simple detection method that helps to reduce the risk of accepting such errors. We study the major histocompatibility complex (MHC) class IIB of guppies, Poecilia reticulata and find that PCR base misincorporations inflate the apparent sequence diversity. When analysing neutral genes, such bias can inflate estimates of effective population size. Previously suggested protocols for identifying genuine alleles are unlikely to exclude all sequencing errors, or they ignore genuine sequence diversity. We present a novel and statistically robust method that reduces the likelihood of accepting PCR artefacts as genuine alleles, and which minimises the necessity of repeated genotyping. Our method identifies sequences that are unlikely to be a PCR artefact, and which need to be independently confirmed through additional PCR of the same template DNA. The proposed methods are recommended particularly for population genetic studies that involve multi-template DNA and in studies on genes with low genetic diversity.

Citation

Cummings, S. M., McMullan, M., Joyce, D. A., & van Oosterhout, C. (2010). Solutions for PCR, cloning and sequencing errors in population genetic analysis. Conservation genetics, 11(3), (1095-1097). doi:10.1007/s10592-009-9864-6. ISSN 1566-0621

Journal Article Type Article
Online Publication Date Mar 1, 2009
Publication Date 2010-06
Journal CONSERVATION GENETICS
Print ISSN 1566-0621
Electronic ISSN 1572-9737
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
Volume 11
Issue 3
Pages 1095-1097
DOI https://doi.org/10.1007/s10592-009-9864-6
Keywords PCR; MHC; guppy
Public URL https://hull-repository.worktribe.com/output/391651
Publisher URL https://link.springer.com/article/10.1007%2Fs10592-009-9864-6