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

Arianna I. Celis; Andrés Aranda-Díaz; Rebecca Culver; Katherine Xue; David Relman; Handuo Shi; Kerwyn Casey Huang

iScience (Apr 2022)

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

Arianna I. Celis,
Andrés Aranda-Díaz,
Rebecca Culver,
Katherine Xue,
David Relman,
Handuo Shi,
Kerwyn Casey Huang

Affiliations

Arianna I. Celis: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
Andrés Aranda-Díaz: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA
Rebecca Culver: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
Katherine Xue: Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
David Relman: Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA
Handuo Shi: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA; Corresponding author
Kerwyn Casey Huang: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Corresponding author

Journal volume & issue: Vol. 25, no. 4
p. 103907

Abstract

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Summary: 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.

Published in iScience

ISSN: 2589-0042 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science
Website: http://www.cell.com/iscience/home

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