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.
References
Acinas SG, Klepac-Ceraj V, Hunt DE, Pharino C, Ceraj I, Distel DL, Polz MF (2004) Fine-scale phylogenetic architecture of a complex bacterial community. Nature 430:551–554. doi:10.1038/nature02649
Acinas SG, Sarma-Rupavtarm R, Klepac-Ceraj V, Polz MF (2005) PCR-induced sequence artifacts and bias: insights from comparison of two 16S rRNA clone libraries constructed from the same sample. Appl Environ Microbiol 71:8966–8969. doi:10.1128/AEM.71.12.8966-8969.2005
Keohavong P, Thilly WG (1989) Fidelity of DNA-polumerases in DNA amplification. Proc Natl Acad Sci USA 86:9253–9257. doi:10.1073/pnas.86.23.9253
Kobayashi N, Tamura K, Aotsuka T (1999) PCR error and molecular population genetics. Biochem Genet 37:317–321. doi:10.1023/A:1018759210666
Lukas D, Vigilant L (2005) Reply: facts, faeces and setting standards for the study of MHC genes using noninvasive samples. Mol Ecol 14:1601–1602. doi:10.1111/j.1365-294X.2005.02459.x
Lukas D, Bradley BJ, Nsubuga AM, Doran-Sheehy D, Robbins MM, Vigilant L (2004) Major histocompatibility complex and microsatellite variation in two populations of wild gorillas. Mol Ecol 13:3389–3402. doi:10.1111/j.1365-294X.2004.02353.x
Thompson JR, Marcelino LA, Polz MF (2002) Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Res 30:2083–2088. doi:10.1093/nar/30.9.2083
van Oosterhout C, Joyce DA, Cummings SM (2006a) Evolution of MHC class IIB in the genome of wild and ornamental guppies, Poecilia reticulata. Heredity 97:111–118. doi:10.1038/sj.hdy.6800843
van Oosterhout C, Joyce DA, Cummings SM, Blais J, Barson NJ, Ramnarine IW, Mohammed RS, Persad N, Cable J (2006b) Balancing selection, random genetic drift, and genetic variation at the major histocompatibility complex in two wild populations of guppies (Poecilia reticulata). Evol Int J Org Evol 60:2562–2574. doi:10.1554/06-286.1
Zylstra P, Rothenfluh HS, Weiller GF, Blanden RV, Steele EJ (1998) PCR amplification of murine immunoglobulin germline V genes: strategies for minimization of recombination artefacts. Immunol Cell Biol 76:395–405. doi:10.1046/j.1440-1711.1998.00772.x
Acknowledgments
We thank Alan Smith for help with fish husbandry. The work was funded by NERC grants NER/M/S/2002/00101, NER/I/S/2000/00885 and Leverhulme Trust grant F/00 181/F awarded to CVO.
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Cummings, S.M., McMullan, M., Joyce, D.A. et al. Solutions for PCR, cloning and sequencing errors in population genetic analysis. Conserv Genet 11, 1095–1097 (2010). https://doi.org/10.1007/s10592-009-9864-6
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DOI: https://doi.org/10.1007/s10592-009-9864-6