Building a Robust 16S Next-Generation Sequencing Analysis Workflow for Complex Microbial Samples

Author(s):

Madelyn A. Szilagyi and Randi M. Pullen

Abstract:

Synthetic microbial communities present an opportunity to expand on current synthetic biology by harnessing bioproduction of Army-relevant materials critical to mission success. Population dynamics in a microbial community change over time, so the quickest way to determine the presence and proportion of any organism in the given community is via 16S sequencing. At ARL, we adapted and refined 16S sequencing workflows to study synthetic microbial communities in laboratory settings. Two model communities were used to validate the workflow: The Hitchhikers of the Rhizosphere (THOR), a simple community with three members, and the Caltech Synthetic Community (Caltech SynCom), a more complex community with 14 organisms. Nucleic acid extraction was identified as a major bottleneck in the process, and while the simpler community was successfully validated, genus-level classification of the complex community remained incomplete, likely due to reliance on pre-trained taxonomic classifiers in bioinformatics analysis. This workflow establishes a framework for future 16S sequencing efforts at ARL, with recommendations to train custom classifiers to ensure confident genus-level identification and support the Army’s biotechnology initiatives.