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 Fanny Packs / Bum Bags 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 Accessories 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.