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

Fonseca VG, Nichols B, Lallias D, Quince C, Carvalho GR, Power DM, Creer S - Nucleic Acids Res. (2012)

Bottom Line: However, estimates of diversity derived from pyrosequencing environmental data sets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification.To further investigate how chimeric molecules are formed, the nSSU gene secondary structure was analyzed in several individuals.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Ecology and Fisheries Genetics Laboratory, School of Biological Sciences, Environment Centre Wales, Bangor University, Deiniol Road, Gwynedd LL57 2UW, UK.

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.

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(a) Nucleotide diversity (Shannon Index) and (b) breakpoint frequencies occurrence in single nematodes and parental chimeric sequences, respectively.
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gks002-F2: (a) Nucleotide diversity (Shannon Index) and (b) breakpoint frequencies occurrence in single nematodes and parental chimeric sequences, respectively.

Mentions: Analysis of chimera breakpoint occurrence in nSSU amplicon sequences revealed that regions with higher nucleotide sequence similarity had significantly higher breakpoint frequencies (P < 0.001, P = 0.00039) (Figure 2a and b). Indeed, in studies with bacteria using the 16S rRNA gene a large number of competing templates with fairly high sequence similarity generated more chimeras (6,16,26). Presumably, one explanation for this phenomenon may be the priming of strand synthesis by prematurely terminated templates in the next PCR round.Figure 2.


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

Fonseca VG, Nichols B, Lallias D, Quince C, Carvalho GR, Power DM, Creer S - Nucleic Acids Res. (2012)

(a) Nucleotide diversity (Shannon Index) and (b) breakpoint frequencies occurrence in single nematodes and parental chimeric sequences, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351157&req=5

gks002-F2: (a) Nucleotide diversity (Shannon Index) and (b) breakpoint frequencies occurrence in single nematodes and parental chimeric sequences, respectively.
Mentions: Analysis of chimera breakpoint occurrence in nSSU amplicon sequences revealed that regions with higher nucleotide sequence similarity had significantly higher breakpoint frequencies (P < 0.001, P = 0.00039) (Figure 2a and b). Indeed, in studies with bacteria using the 16S rRNA gene a large number of competing templates with fairly high sequence similarity generated more chimeras (6,16,26). Presumably, one explanation for this phenomenon may be the priming of strand synthesis by prematurely terminated templates in the next PCR round.Figure 2.

Bottom Line: However, estimates of diversity derived from pyrosequencing environmental data sets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification.To further investigate how chimeric molecules are formed, the nSSU gene secondary structure was analyzed in several individuals.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Ecology and Fisheries Genetics Laboratory, School of Biological Sciences, Environment Centre Wales, Bangor University, Deiniol Road, Gwynedd LL57 2UW, UK.

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.

Show MeSH
Related in: MedlinePlus