Cargando…

Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals

While microbial communities inhabit a wide variety of complex natural environments, in vitro culturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights. In vitro assemblies of gut commensals have recently been introduced as models for the int...

Descripción completa

Detalles Bibliográficos
Autores principales: Celis, Arianna I., Aranda-Díaz, Andrés, Culver, Rebecca, Xue, Katherine, Relman, David, Shi, Handuo, Huang, Kerwyn Casey
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8941205/
https://www.ncbi.nlm.nih.gov/pubmed/35340431
http://dx.doi.org/10.1016/j.isci.2022.103907
_version_ 1784673059911761920
author Celis, Arianna I.
Aranda-Díaz, Andrés
Culver, Rebecca
Xue, Katherine
Relman, David
Shi, Handuo
Huang, Kerwyn Casey
author_facet Celis, Arianna I.
Aranda-Díaz, Andrés
Culver, Rebecca
Xue, Katherine
Relman, David
Shi, Handuo
Huang, Kerwyn Casey
author_sort Celis, Arianna I.
collection PubMed
description While microbial communities inhabit a wide variety of complex natural environments, in vitro culturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights. In vitro assemblies of gut commensals have recently been introduced as models for the intestinal microbiota, which plays fundamental roles in host health. However, a protocol for 16S rRNA sequencing and analysis of in vitro samples that optimizes financial cost, time/effort, and accuracy/reproducibility has yet to be established. Here, we systematically identify protocol elements that have significant impact, introduce bias, and/or can be simplified. Our results indicate that community diversity and composition are generally unaffected by substantial protocol streamlining. Additionally, we demonstrate that a strictly aerobic halophile is an effective spike-in for estimating absolute abundances in communities of anaerobic gut commensals. This time- and money-saving protocol should accelerate discovery by increasing 16S rRNA data reliability and comparability and through the incorporation of absolute abundance estimates.
format Online
Article
Text
id pubmed-8941205
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Elsevier
record_format MEDLINE/PubMed
spelling pubmed-89412052022-03-24 Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals Celis, Arianna I. Aranda-Díaz, Andrés Culver, Rebecca Xue, Katherine Relman, David Shi, Handuo Huang, Kerwyn Casey iScience Article While microbial communities inhabit a wide variety of complex natural environments, in vitro culturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights. In vitro assemblies of gut commensals have recently been introduced as models for the intestinal microbiota, which plays fundamental roles in host health. However, a protocol for 16S rRNA sequencing and analysis of in vitro samples that optimizes financial cost, time/effort, and accuracy/reproducibility has yet to be established. Here, we systematically identify protocol elements that have significant impact, introduce bias, and/or can be simplified. Our results indicate that community diversity and composition are generally unaffected by substantial protocol streamlining. Additionally, we demonstrate that a strictly aerobic halophile is an effective spike-in for estimating absolute abundances in communities of anaerobic gut commensals. This time- and money-saving protocol should accelerate discovery by increasing 16S rRNA data reliability and comparability and through the incorporation of absolute abundance estimates. Elsevier 2022-02-11 /pmc/articles/PMC8941205/ /pubmed/35340431 http://dx.doi.org/10.1016/j.isci.2022.103907 Text en © 2022. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Celis, Arianna I.
Aranda-Díaz, Andrés
Culver, Rebecca
Xue, Katherine
Relman, David
Shi, Handuo
Huang, Kerwyn Casey
Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals
title Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals
title_full Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals
title_fullStr Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals
title_full_unstemmed Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals
title_short Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals
title_sort optimization of the 16s rrna sequencing analysis pipeline for studying in vitro communities of gut commensals
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8941205/
https://www.ncbi.nlm.nih.gov/pubmed/35340431
http://dx.doi.org/10.1016/j.isci.2022.103907
work_keys_str_mv AT celisariannai optimizationofthe16srrnasequencinganalysispipelineforstudyinginvitrocommunitiesofgutcommensals
AT arandadiazandres optimizationofthe16srrnasequencinganalysispipelineforstudyinginvitrocommunitiesofgutcommensals
AT culverrebecca optimizationofthe16srrnasequencinganalysispipelineforstudyinginvitrocommunitiesofgutcommensals
AT xuekatherine optimizationofthe16srrnasequencinganalysispipelineforstudyinginvitrocommunitiesofgutcommensals
AT relmandavid optimizationofthe16srrnasequencinganalysispipelineforstudyinginvitrocommunitiesofgutcommensals
AT shihanduo optimizationofthe16srrnasequencinganalysispipelineforstudyinginvitrocommunitiesofgutcommensals
AT huangkerwyncasey optimizationofthe16srrnasequencinganalysispipelineforstudyinginvitrocommunitiesofgutcommensals