Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses

Fonseca, V.G., Nichols, B., Lallias, D., Quince, C., Carvalho, G.R., Power, D.M. and Creer, S. (2012) Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses. Nucleic Acids Research, 40(9), e66. (doi: 10.1093/nar/gks002)

Abstract

Eukaryotic diversity in environmental samples is often assessed via PCR-based amplification of nSSU genes. However, estimates of diversity derived from pyrosequencing environmental data sets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification. Chimeras are hybrid products composed of distinct parental sequences that can lead to the misinterpretation of diversity estimates. We have analyzed the effect of sample richness, evenness and phylogenetic diversity on the formation of chimeras using a nSSU data set derived from 454 Roche pyrosequencing of replicated, large control pools of closely and distantly related nematode mock communities, of known intragenomic identity and richness. To further investigate how chimeric molecules are formed, the nSSU gene secondary structure was analyzed in several individuals. For the first time in eukaryotes, chimera formation proved to be higher in both richer and more genetically diverse samples, thus providing a novel perspective of chimera formation in pyrosequenced environmental data sets. Findings contribute to a better understanding of the nature and mechanisms involved in chimera formation during PCR amplification of environmentally derived DNA. Moreover, given the similarities between biodiversity analyses using amplicon sequencing and those used to assess genomic variation, our findings have potential broad application for identifying genetic variation in homologous loci or multigene families in general.<p></p>

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Quince, Dr Christopher
• Authors: Fonseca, V.G., Nichols, B., Lallias, D., Quince, C., Carvalho, G.R., Power, D.M., and Creer, S.
• College/School: College of Science and Engineering > School of Engineering > Infrastructure and Environment
• Journal Name: Nucleic Acids Research
• Publisher: Oxford University Press
• ISSN: 0305-1048
• ISSN (Online): 1362-4962
• Published Online: 25 January 2012
• Copyright Holders: Copyright © 2012 The Authors
• First Published: First published in Nucleic Acids Research 40(9):e66
• Publisher Policy: Reproduced under a Creative Commons License

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
50335	1	Pioneering the genomics era of environmental microbiology	Christopher Quince	Engineering & Physical Sciences Research Council (EPSRC)	EP/H003851/1	Infrastructure and Environment