Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations

ABSTRACT High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly...

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Autores principales:	Kasia Piwosz, Tanja Shabarova, Jakob Pernthaler, Thomas Posch, Karel Šimek, Petr Porcal, Michaela M. Salcher
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	CARD-FISH amplicon sequencing bacterial communities bacterial community structure bacterial dynamics eukaryotic communities Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d5af1d57525746b3b8280c7880ab35dd
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spelling	oai:doaj.org-article:d5af1d57525746b3b8280c7880ab35dd2021-11-15T15:29:17ZBacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations10.1128/mSphere.00052-202379-5042https://doaj.org/article/d5af1d57525746b3b8280c7880ab35dd2020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00052-20https://doaj.org/toc/2379-5042ABSTRACT High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series (n = 883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence in situ hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards. IMPORTANCE High-throughput sequencing (HTS) of amplified fragments of rRNA genes provides unprecedented insight into the diversity of prokaryotic and eukaryotic microorganisms. Unfortunately, HTS data are prone to quantitative biases, which may lead to an erroneous picture of microbial community composition and thwart efforts to advance its understanding. These concerns motivated us to investigate how accurately HTS data characterize the variability of microbial communities, the relative abundances of specific phylotypes, and their relationships with environmental factors in comparison to an established microscopy-based method. We compared results obtained by HTS and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) from three independent aquatic time series for both prokaryotic and eukaryotic microorganisms (almost 900 data points, the largest obtained with both methods so far). HTS and CARD-FISH data disagree with regard to relative abundances of bacterial and eukaryotic phylotypes but identify similar environmental drivers shaping bacterial and eukaryotic communities.Kasia PiwoszTanja ShabarovaJakob PernthalerThomas PoschKarel ŠimekPetr PorcalMichaela M. SalcherAmerican Society for MicrobiologyarticleCARD-FISHamplicon sequencingbacterial communitiesbacterial community structurebacterial dynamicseukaryotic communitiesMicrobiologyQR1-502ENmSphere, Vol 5, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	CARD-FISH amplicon sequencing bacterial communities bacterial community structure bacterial dynamics eukaryotic communities Microbiology QR1-502
spellingShingle	CARD-FISH amplicon sequencing bacterial communities bacterial community structure bacterial dynamics eukaryotic communities Microbiology QR1-502 Kasia Piwosz Tanja Shabarova Jakob Pernthaler Thomas Posch Karel Šimek Petr Porcal Michaela M. Salcher Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations
description	ABSTRACT High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series (n = 883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence in situ hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards. IMPORTANCE High-throughput sequencing (HTS) of amplified fragments of rRNA genes provides unprecedented insight into the diversity of prokaryotic and eukaryotic microorganisms. Unfortunately, HTS data are prone to quantitative biases, which may lead to an erroneous picture of microbial community composition and thwart efforts to advance its understanding. These concerns motivated us to investigate how accurately HTS data characterize the variability of microbial communities, the relative abundances of specific phylotypes, and their relationships with environmental factors in comparison to an established microscopy-based method. We compared results obtained by HTS and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) from three independent aquatic time series for both prokaryotic and eukaryotic microorganisms (almost 900 data points, the largest obtained with both methods so far). HTS and CARD-FISH data disagree with regard to relative abundances of bacterial and eukaryotic phylotypes but identify similar environmental drivers shaping bacterial and eukaryotic communities.
format	article
author	Kasia Piwosz Tanja Shabarova Jakob Pernthaler Thomas Posch Karel Šimek Petr Porcal Michaela M. Salcher
author_facet	Kasia Piwosz Tanja Shabarova Jakob Pernthaler Thomas Posch Karel Šimek Petr Porcal Michaela M. Salcher
author_sort	Kasia Piwosz
title	Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations
title_short	Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations
title_full	Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations
title_fullStr	Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations
title_full_unstemmed	Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations
title_sort	bacterial and eukaryotic small-subunit amplicon data do not provide a quantitative picture of microbial communities, but they are reliable in the context of ecological interpretations
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/d5af1d57525746b3b8280c7880ab35dd
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Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations

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