Genome-based targeted sequencing as a reproducible microbial community profiling assay.

Abstract

Current sequencing-based methods for profiling microbial communities rely on marker gene (e.g. 16S rRNA) or metagenome shotgun sequencing (mWGS) analysis. We present a new approach based on highly multiplexed oligonucleotide probes designed from reference genomes in a pooled primer-extension reaction during library construction to derive relative abundance data. This approach, termed MA-GenTA: Microbial Abundances from Genome Tagged Analysis, enables quantitative, straightforward, cost-effective microbiome profiling that combines desirable features of both 16S rRNA and mWGS strategies. To test the utility of the MA-GenTA assay, probes were designed for 830 genome sequences representing bacteria present in mouse stool specimens. Comparison of the MA-GenTA data with mWGS data demonstrated excellent correlation down to 0.01% relative abundance and a similar number of organisms detected per sample. Despite the incompleteness of the reference database, NMDS clustering based on the Bray-Curtis dissimilarity metric of sample groups was consistent between MA-GenTA, mWGS and 16S rRNA datasets. MA-GenTA represents a potentially useful new method for microbiome community profiling based on reference genomes.