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Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection

INTRODUCTION: Accurate analyses of microbiota composition of low-density communities (10(3)–10(4) bacteria/sample) can be challenging. Background DNA from chemicals and consumables, extraction biases as well as differences in PCR efficiency can significantly interfere with microbiota assessment. Thi...

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Autores principales: Biesbroek, Giske, Sanders, Elisabeth A. M., Roeselers, Guus, Wang, Xinhui, Caspers, Martien P. M., Trzciński, Krzysztof, Bogaert, Debby, Keijser, Bart J. F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295791/
https://www.ncbi.nlm.nih.gov/pubmed/22412957
http://dx.doi.org/10.1371/journal.pone.0032942
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author Biesbroek, Giske
Sanders, Elisabeth A. M.
Roeselers, Guus
Wang, Xinhui
Caspers, Martien P. M.
Trzciński, Krzysztof
Bogaert, Debby
Keijser, Bart J. F.
author_facet Biesbroek, Giske
Sanders, Elisabeth A. M.
Roeselers, Guus
Wang, Xinhui
Caspers, Martien P. M.
Trzciński, Krzysztof
Bogaert, Debby
Keijser, Bart J. F.
author_sort Biesbroek, Giske
collection PubMed
description INTRODUCTION: Accurate analyses of microbiota composition of low-density communities (10(3)–10(4) bacteria/sample) can be challenging. Background DNA from chemicals and consumables, extraction biases as well as differences in PCR efficiency can significantly interfere with microbiota assessment. This study was aiming to establish protocols for accurate microbiota analysis at low microbial density. METHODS: To examine possible effects of bacterial density on microbiota analyses we compared microbiota profiles of serial diluted saliva and low (nares, nasopharynx) and high-density (oropharynx) upper airway communities in four healthy individuals. DNA was extracted with four different extraction methods (Epicentre Masterpure, Qiagen DNeasy, Mobio Powersoil and a phenol bead-beating protocol combined with Agowa-Mag-mini). Bacterial DNA recovery was analysed by 16S qPCR and microbiota profiles through GS-FLX-Titanium-Sequencing of 16S rRNA gene amplicons spanning the V5–V7 regions. RESULTS: Lower template concentrations significantly impacted microbiota profiling results. With higher dilutions, low abundant species were overrepresented. In samples of <10(5) bacteria per ml, e.g. DNA <1 pg/µl, microbiota profiling deviated from the original sample and other dilutions showing a significant increase in the taxa Proteobacteria and decrease in Bacteroidetes. In similar low density samples, DNA extraction method determined if DNA levels were below or above 1 pg/µl and, together with lysis preferences per method, had profound impact on microbiota analyses in both relative abundance as well as representation of species. CONCLUSION: This study aimed to interpret microbiota analyses of low-density communities. Bacterial density seemed to interfere with microbiota analyses at < than 10(6) bacteria per ml or DNA <1 pg/µl. We therefore recommend this threshold for working with low density materials. This study underlines that bias reduction is crucial for adequate profiling of especially low-density bacterial communities.
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spelling pubmed-32957912012-03-12 Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection Biesbroek, Giske Sanders, Elisabeth A. M. Roeselers, Guus Wang, Xinhui Caspers, Martien P. M. Trzciński, Krzysztof Bogaert, Debby Keijser, Bart J. F. PLoS One Research Article INTRODUCTION: Accurate analyses of microbiota composition of low-density communities (10(3)–10(4) bacteria/sample) can be challenging. Background DNA from chemicals and consumables, extraction biases as well as differences in PCR efficiency can significantly interfere with microbiota assessment. This study was aiming to establish protocols for accurate microbiota analysis at low microbial density. METHODS: To examine possible effects of bacterial density on microbiota analyses we compared microbiota profiles of serial diluted saliva and low (nares, nasopharynx) and high-density (oropharynx) upper airway communities in four healthy individuals. DNA was extracted with four different extraction methods (Epicentre Masterpure, Qiagen DNeasy, Mobio Powersoil and a phenol bead-beating protocol combined with Agowa-Mag-mini). Bacterial DNA recovery was analysed by 16S qPCR and microbiota profiles through GS-FLX-Titanium-Sequencing of 16S rRNA gene amplicons spanning the V5–V7 regions. RESULTS: Lower template concentrations significantly impacted microbiota profiling results. With higher dilutions, low abundant species were overrepresented. In samples of <10(5) bacteria per ml, e.g. DNA <1 pg/µl, microbiota profiling deviated from the original sample and other dilutions showing a significant increase in the taxa Proteobacteria and decrease in Bacteroidetes. In similar low density samples, DNA extraction method determined if DNA levels were below or above 1 pg/µl and, together with lysis preferences per method, had profound impact on microbiota analyses in both relative abundance as well as representation of species. CONCLUSION: This study aimed to interpret microbiota analyses of low-density communities. Bacterial density seemed to interfere with microbiota analyses at < than 10(6) bacteria per ml or DNA <1 pg/µl. We therefore recommend this threshold for working with low density materials. This study underlines that bias reduction is crucial for adequate profiling of especially low-density bacterial communities. Public Library of Science 2012-03-06 /pmc/articles/PMC3295791/ /pubmed/22412957 http://dx.doi.org/10.1371/journal.pone.0032942 Text en Biesbroek 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
Biesbroek, Giske
Sanders, Elisabeth A. M.
Roeselers, Guus
Wang, Xinhui
Caspers, Martien P. M.
Trzciński, Krzysztof
Bogaert, Debby
Keijser, Bart J. F.
Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection
title Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection
title_full Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection
title_fullStr Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection
title_full_unstemmed Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection
title_short Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection
title_sort deep sequencing analyses of low density microbial communities: working at the boundary of accurate microbiota detection
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295791/
https://www.ncbi.nlm.nih.gov/pubmed/22412957
http://dx.doi.org/10.1371/journal.pone.0032942
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