Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.

Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Her...

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Autores principales:	Dana Willner, Joshua Daly, David Whiley, Keith Grimwood, Claire E Wainwright, Philip Hugenholtz
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1c2e1c37701c4b1c95baa65ef79214a5
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spelling	oai:doaj.org-article:1c2e1c37701c4b1c95baa65ef79214a52021-11-18T07:22:18ZComparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.1932-620310.1371/journal.pone.0034605https://doaj.org/article/1c2e1c37701c4b1c95baa65ef79214a52012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22514642/?tool=EBIhttps://doaj.org/toc/1932-6203Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Here we assessed five different extraction methods on pediatric bronchoalveolar lavage (BAL) samples and a mock community comprised of nine bacterial genera to determine method reproducibility and detection limits for these typically low complexity communities. Additionally, using the mock community, we were able to evaluate contamination and select a relative abundance cut-off threshold based on the geometric distribution that optimizes the trade off between detecting bona fide operational taxonomic units and filtering out spurious ones. Using this threshold, the majority of genera in the mock community were predictably detected by all extraction methods including the hard-to-lyse Gram-positive genus Staphylococcus. Differences between extraction methods were significantly greater than between technical replicates for both the mock community and BAL samples emphasizing the importance of using a standardized methodology for microbiome studies. However, regardless of method used, individual patients retained unique diagnostic profiles. Furthermore, despite being stored as raw frozen samples for over five years, community profiles from BAL samples were consistent with historical culturing results. The culture-independent profiling of these samples also identified a number of anaerobic genera that are gaining acceptance as being part of the respiratory microbiome. This study should help guide researchers to formulate sampling, extraction and analysis strategies for respiratory and other human microbiome samples.Dana WillnerJoshua DalyDavid WhileyKeith GrimwoodClaire E WainwrightPhilip HugenholtzPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 4, p e34605 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Dana Willner Joshua Daly David Whiley Keith Grimwood Claire E Wainwright Philip Hugenholtz Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.
description	Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Here we assessed five different extraction methods on pediatric bronchoalveolar lavage (BAL) samples and a mock community comprised of nine bacterial genera to determine method reproducibility and detection limits for these typically low complexity communities. Additionally, using the mock community, we were able to evaluate contamination and select a relative abundance cut-off threshold based on the geometric distribution that optimizes the trade off between detecting bona fide operational taxonomic units and filtering out spurious ones. Using this threshold, the majority of genera in the mock community were predictably detected by all extraction methods including the hard-to-lyse Gram-positive genus Staphylococcus. Differences between extraction methods were significantly greater than between technical replicates for both the mock community and BAL samples emphasizing the importance of using a standardized methodology for microbiome studies. However, regardless of method used, individual patients retained unique diagnostic profiles. Furthermore, despite being stored as raw frozen samples for over five years, community profiles from BAL samples were consistent with historical culturing results. The culture-independent profiling of these samples also identified a number of anaerobic genera that are gaining acceptance as being part of the respiratory microbiome. This study should help guide researchers to formulate sampling, extraction and analysis strategies for respiratory and other human microbiome samples.
format	article
author	Dana Willner Joshua Daly David Whiley Keith Grimwood Claire E Wainwright Philip Hugenholtz
author_facet	Dana Willner Joshua Daly David Whiley Keith Grimwood Claire E Wainwright Philip Hugenholtz
author_sort	Dana Willner
title	Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.
title_short	Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.
title_full	Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.
title_fullStr	Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.
title_full_unstemmed	Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.
title_sort	comparison of dna extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/1c2e1c37701c4b1c95baa65ef79214a5
work_keys_str_mv	AT danawillner comparisonofdnaextractionmethodsformicrobialcommunityprofilingwithanapplicationtopediatricbronchoalveolarlavagesamples AT joshuadaly comparisonofdnaextractionmethodsformicrobialcommunityprofilingwithanapplicationtopediatricbronchoalveolarlavagesamples AT davidwhiley comparisonofdnaextractionmethodsformicrobialcommunityprofilingwithanapplicationtopediatricbronchoalveolarlavagesamples AT keithgrimwood comparisonofdnaextractionmethodsformicrobialcommunityprofilingwithanapplicationtopediatricbronchoalveolarlavagesamples AT claireewainwright comparisonofdnaextractionmethodsformicrobialcommunityprofilingwithanapplicationtopediatricbronchoalveolarlavagesamples AT philiphugenholtz comparisonofdnaextractionmethodsformicrobialcommunityprofilingwithanapplicationtopediatricbronchoalveolarlavagesamples
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Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.

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