摘要：SummaryWhile microbial communities inhabit a wide variety of complex natural environments,in vitroculturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights.In vitroassemblies 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 ofin vitrosamples 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.Graphical abstractDisplay OmittedHighlights•Systematic optimization of 16S library generation from gutin vitrocommunities•Community diversity and composition are generally reproducible after streamlining•Halomonas elongatais an effective spike-in for estimating absolute abundance•This time- and money-saving protocol should accelerate discoveryMicrobiology; Methodology in biological sciences
