Cargando…

Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing

Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have inves...

Descripción completa

Detalles Bibliográficos
Autores principales: Lee, Charles K., Herbold, Craig W., Polson, Shawn W., Wommack, K. Eric, Williamson, Shannon J., McDonald, Ian R., Cary, S. Craig
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435322/
https://www.ncbi.nlm.nih.gov/pubmed/22970184
http://dx.doi.org/10.1371/journal.pone.0044224
_version_ 1782242511181316096
author Lee, Charles K.
Herbold, Craig W.
Polson, Shawn W.
Wommack, K. Eric
Williamson, Shannon J.
McDonald, Ian R.
Cary, S. Craig
author_facet Lee, Charles K.
Herbold, Craig W.
Polson, Shawn W.
Wommack, K. Eric
Williamson, Shannon J.
McDonald, Ian R.
Cary, S. Craig
author_sort Lee, Charles K.
collection PubMed
description Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3–V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.
format Online
Article
Text
id pubmed-3435322
institution National Center for Biotechnology Information
language English
publishDate 2012
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-34353222012-09-11 Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing Lee, Charles K. Herbold, Craig W. Polson, Shawn W. Wommack, K. Eric Williamson, Shannon J. McDonald, Ian R. Cary, S. Craig PLoS One Research Article Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3–V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure. Public Library of Science 2012-09-06 /pmc/articles/PMC3435322/ /pubmed/22970184 http://dx.doi.org/10.1371/journal.pone.0044224 Text en © 2012 Lee et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Lee, Charles K.
Herbold, Craig W.
Polson, Shawn W.
Wommack, K. Eric
Williamson, Shannon J.
McDonald, Ian R.
Cary, S. Craig
Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing
title Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing
title_full Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing
title_fullStr Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing
title_full_unstemmed Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing
title_short Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing
title_sort groundtruthing next-gen sequencing for microbial ecology–biases and errors in community structure estimates from pcr amplicon pyrosequencing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435322/
https://www.ncbi.nlm.nih.gov/pubmed/22970184
http://dx.doi.org/10.1371/journal.pone.0044224
work_keys_str_mv AT leecharlesk groundtruthingnextgensequencingformicrobialecologybiasesanderrorsincommunitystructureestimatesfrompcrampliconpyrosequencing
AT herboldcraigw groundtruthingnextgensequencingformicrobialecologybiasesanderrorsincommunitystructureestimatesfrompcrampliconpyrosequencing
AT polsonshawnw groundtruthingnextgensequencingformicrobialecologybiasesanderrorsincommunitystructureestimatesfrompcrampliconpyrosequencing
AT wommackkeric groundtruthingnextgensequencingformicrobialecologybiasesanderrorsincommunitystructureestimatesfrompcrampliconpyrosequencing
AT williamsonshannonj groundtruthingnextgensequencingformicrobialecologybiasesanderrorsincommunitystructureestimatesfrompcrampliconpyrosequencing
AT mcdonaldianr groundtruthingnextgensequencingformicrobialecologybiasesanderrorsincommunitystructureestimatesfrompcrampliconpyrosequencing
AT caryscraig groundtruthingnextgensequencingformicrobialecologybiasesanderrorsincommunitystructureestimatesfrompcrampliconpyrosequencing