Limits...
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.

Show MeSH

Related in: MedlinePlus

Most frequent predicted secondary structures found on the 18S rDNA amplicon at (a) 55°C and (b) 65°C folding temperatures, using as an example two single nematodes. Arrows indicate where the most frequent breakpoints occur generally matching hairpin-loops. 79TN11 consensus and 25LBFB8F04 indicate the labels given for each nematode. dG: free energy necessary for sequence stability at a given temperature.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351157&req=5

gks002-F3: Most frequent predicted secondary structures found on the 18S rDNA amplicon at (a) 55°C and (b) 65°C folding temperatures, using as an example two single nematodes. Arrows indicate where the most frequent breakpoints occur generally matching hairpin-loops. 79TN11 consensus and 25LBFB8F04 indicate the labels given for each nematode. dG: free energy necessary for sequence stability at a given temperature.

Mentions: Chimeras are generally composed of two true sequences, occasionally more (8), with a discrete break point where the transition from one sequence to another occurs. In the present data set, the distribution of chimera breakpoints showed a similar pattern across closely and distantly related nematode assemblages, with a mean peak of frequency at the first 140 bp of the selected nSSU region (Supplementary Figure S1). Although GC content is thought to correlate with chimera formation due to inefficient strand separation and susceptibility to secondary structure formation, a detailed analysis of the parent chimeric sequences at the breakpoints did not reveal a significant correlation between GC rich regions and chimera frequencies. To further investigate the breakpoint region, the secondary structure of the amplified nSSU fragment was modeled in 12 single nematode sequences at 55°C and 65°C folding temperatures (Supplementary Table S1). Analysis of the nSSU secondary structure showed that the regions where the breakpoints occurred coincided with hairpin loop structures at both temperatures, although at 65°C regions of secondary structure were less abundant (Supplementary Table S1 and Figure 3).Figure 3.


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)

Most frequent predicted secondary structures found on the 18S rDNA amplicon at (a) 55°C and (b) 65°C folding temperatures, using as an example two single nematodes. Arrows indicate where the most frequent breakpoints occur generally matching hairpin-loops. 79TN11 consensus and 25LBFB8F04 indicate the labels given for each nematode. dG: free energy necessary for sequence stability at a given temperature.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks002-F3: Most frequent predicted secondary structures found on the 18S rDNA amplicon at (a) 55°C and (b) 65°C folding temperatures, using as an example two single nematodes. Arrows indicate where the most frequent breakpoints occur generally matching hairpin-loops. 79TN11 consensus and 25LBFB8F04 indicate the labels given for each nematode. dG: free energy necessary for sequence stability at a given temperature.
Mentions: Chimeras are generally composed of two true sequences, occasionally more (8), with a discrete break point where the transition from one sequence to another occurs. In the present data set, the distribution of chimera breakpoints showed a similar pattern across closely and distantly related nematode assemblages, with a mean peak of frequency at the first 140 bp of the selected nSSU region (Supplementary Figure S1). Although GC content is thought to correlate with chimera formation due to inefficient strand separation and susceptibility to secondary structure formation, a detailed analysis of the parent chimeric sequences at the breakpoints did not reveal a significant correlation between GC rich regions and chimera frequencies. To further investigate the breakpoint region, the secondary structure of the amplified nSSU fragment was modeled in 12 single nematode sequences at 55°C and 65°C folding temperatures (Supplementary Table S1). Analysis of the nSSU secondary structure showed that the regions where the breakpoints occurred coincided with hairpin loop structures at both temperatures, although at 65°C regions of secondary structure were less abundant (Supplementary Table S1 and Figure 3).Figure 3.

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