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 an approach based on a single-primer extension reaction using a highly multiplexed oligonucleotide probe pool. 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. The use of multiple probes per target genome and rigorous probe design criteria enabled robust determination of relative abundance. 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, nonmetric multidimensional scaling (NMDS) clustering based on the Bray-Curtis dissimilarity metric of sample groups was consistent between MA-GenTA, mWGS, and 16S rRNA data sets. MA-GenTA represents a potentially useful new method for microbiome community profiling based on reference genomes. IMPORTANCE New methods for profiling the microbial communities can create new approaches to understanding the composition and function of those communities. In this study, we combined bacterial genome-specific probe design with a highly multiplexed single primer extension reaction as a new method to profile microbial communities, using stool from various mouse strains as a test case. This method, termed MA-GenTA, was benchmarked against 16S rRNA gene sequencing and metagenome sequencing methods and delivered similar relative abundance and clustering data. Since the probes were generated from reference genomes, MA-GenTA was also able to provide functional pathway data for the stool microbiome in the assayed samples. The method is more informative than 16S rRNA analysis while being less costly than metagenome shotgun sequencing.

Current Uses and Future Perspectives of Genomic Technologies in Clinical Microbiology

“…In microbial identification, tNGS is particularly valuable for distinguishing bacterial species using the 16S ribosomal RNA (rRNA) gene or fungal species through the internal transcribed spacer 1/2 (ITS-1/2) regions. This approach enables precise taxonomic classification and can be applied to various environmental samples, clinical specimens, and food safety assessments [53][54][55]. However, one substantial challenge in tNGS is the meticulous selection of target regions.…”

Development of a high-density sub-species-specific targeted SNP assay for Rocky Mountain bighorn sheep (Ovis canadensis canadensis)

“…This form of SPET uses a simplified single primer design and functions by sequencing flanking regions around a probe to sequence target regions of interest ( Barchi et al, 2019 ). Despite being a relatively new technology, it has proven applications in humans ( Nairismägi et al, 2016 ; Saber et al, 2017 ; Scolnick et al, 2015 ), non-human mammals ( Andrews et al, 2021 ; Gavriliuc et al, 2022 ), plants ( Barchi et al, 2019 ; Gramazio et al, 2020 ; Scaglione et al, 2019 ), arthropods ( Chang et al, 2020 ; Vu et al, 2023 ), and bacteria ( Benjamino et al, 2021 ; Homeier-Bachmann et al, 2022 ).…”