Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals

16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given th...

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Autores principales:	Hannah E. Epstein, Alejandra Hernandez-Agreda, Samuel Starko, Julia K. Baum, Rebecca Vega Thurber
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	amplicon sequencing 16S rRNA protocol comparison coral microbiome microbial diversity Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/67aa2c74754646f99fc5282c7cd46e99
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spelling	oai:doaj.org-article:67aa2c74754646f99fc5282c7cd46e992021-12-01T01:47:54ZInconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals1664-302X10.3389/fmicb.2021.740932https://doaj.org/article/67aa2c74754646f99fc5282c7cd46e992021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmicb.2021.740932/fullhttps://doaj.org/toc/1664-302X16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given that the same variable region of the 16S gene is targeted, it is generally accepted that sequencing output from differing protocols are comparable and this assumption underlies our ability to identify universal patterns in microbial dynamics through meta-analyses. However, discrepant results from a combined 16S rRNA gene dataset prepared by two labs whose protocols differed only in DNA polymerase and sequencing platform led us to scrutinize the outputs and challenge the idea of confidently combining them for standard microbiome analysis. Using technical replicates of reef-building coral samples from two species, Montipora aequituberculata and Porites lobata, we evaluated the consistency of alpha and beta diversity metrics between data resulting from these highly similar protocols. While we found minimal variation in alpha diversity between platform, significant differences were revealed with most beta diversity metrics, dependent on host species. These inconsistencies persisted following removal of low abundance taxa and when comparing across higher taxonomic levels, suggesting that bacterial community differences associated with sequencing protocol are likely to be context dependent and difficult to correct without extensive validation work. The results of this study encourage caution in the statistical comparison and interpretation of studies that combine rRNA gene sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences.Hannah E. EpsteinAlejandra Hernandez-AgredaSamuel StarkoJulia K. BaumRebecca Vega ThurberFrontiers Media S.A.articleamplicon sequencing16S rRNAprotocol comparisoncoral microbiomemicrobial diversityMicrobiologyQR1-502ENFrontiers in Microbiology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	amplicon sequencing 16S rRNA protocol comparison coral microbiome microbial diversity Microbiology QR1-502
spellingShingle	amplicon sequencing 16S rRNA protocol comparison coral microbiome microbial diversity Microbiology QR1-502 Hannah E. Epstein Alejandra Hernandez-Agreda Samuel Starko Julia K. Baum Rebecca Vega Thurber Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals
description	16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given that the same variable region of the 16S gene is targeted, it is generally accepted that sequencing output from differing protocols are comparable and this assumption underlies our ability to identify universal patterns in microbial dynamics through meta-analyses. However, discrepant results from a combined 16S rRNA gene dataset prepared by two labs whose protocols differed only in DNA polymerase and sequencing platform led us to scrutinize the outputs and challenge the idea of confidently combining them for standard microbiome analysis. Using technical replicates of reef-building coral samples from two species, Montipora aequituberculata and Porites lobata, we evaluated the consistency of alpha and beta diversity metrics between data resulting from these highly similar protocols. While we found minimal variation in alpha diversity between platform, significant differences were revealed with most beta diversity metrics, dependent on host species. These inconsistencies persisted following removal of low abundance taxa and when comparing across higher taxonomic levels, suggesting that bacterial community differences associated with sequencing protocol are likely to be context dependent and difficult to correct without extensive validation work. The results of this study encourage caution in the statistical comparison and interpretation of studies that combine rRNA gene sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences.
format	article
author	Hannah E. Epstein Alejandra Hernandez-Agreda Samuel Starko Julia K. Baum Rebecca Vega Thurber
author_facet	Hannah E. Epstein Alejandra Hernandez-Agreda Samuel Starko Julia K. Baum Rebecca Vega Thurber
author_sort	Hannah E. Epstein
title	Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals
title_short	Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals
title_full	Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals
title_fullStr	Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals
title_full_unstemmed	Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals
title_sort	inconsistent patterns of microbial diversity and composition between highly similar sequencing protocols: a case study with reef-building corals
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/67aa2c74754646f99fc5282c7cd46e99
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Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals

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