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
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.
