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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2022
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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 |
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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 |
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