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

Cummings, SM, McMullan, M, Joyce, DA and Van Oosterhout, C (2010) Solutions for PCR, cloning and sequencing errors in population genetic analysis. Conservation Genetics, 11 (3). pp. 1095-1097.

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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.

Item Type: Article
Faculty \ School: Faculty of Science > School of Environmental Sciences
Depositing User: Rachel Snow
Date Deposited: 24 Feb 2011 15:27
Last Modified: 24 Jul 2019 14:03
URI: https://ueaeprints.uea.ac.uk/id/eprint/24647
DOI: 10.1007/s10592-009-9864-6

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