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Publication
Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses
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Advisor(s)
Abstract(s)
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.
Description
Keywords
Citation
V. G. Fonseca, B. Nichols, D. Lallias, C. Quince, G. R. Carvalho, D. M. Power and S. Creer, "Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyse" in Nucleic Acids Research, 2012, 1–9.
Publisher
Oxford University Press