Abstract 1484: Microbial sequencing using a single-pool target enrichment of multiple variable regions of the 16S rRNA gene, the nuclear ribosomal internal transcribed spacer (ITS) region, and antimicrobial resistance genes
Authors: Peck Kayla , Stedtfeld Robert , RoseFigura Jordan , Reed Brett , McUsic Drew , Irish Jon , Etchebarne Brett , Johnson Timothy , Kurihara Laurie , Makarov Vladimir 
Published: August 2019

The human microbiome has been studied in the context of a number of diseases and has recently been gaining momentum in the field of cancer research. The composition of the gut microbiome, for example, has been shown to affect cancer progression and may act as both a biomarker and therapeutic target. Metagenomic shotgun sequencing has played a large role in characterizing the composition of microbial communities. However, direct sequencing of multiple complex samples in parallel has relatively low sensitivity and struggles to accurately characterize the microbiome. Alternatively, targeted PCR and sequencing can be used to simultaneously enrich for and identify multiple microbial targets of interest in parallel. Here, we present a single-pool multiplex PCR assay to enrich for multiple variable regions of the 16S ribosomal RNA (rRNA) gene, the nuclear ribosomal internal transcribed spacer (ITS) region, and 35 antibiotic resistance genes from multiple bacteria species listed as threats by the CDC. Our two-hour protocol generates target-enriched libraries from genomic DNA (gDNA) that are compatible with Illumina sequencing platforms. We validated our protocol using gDNA from mock microbial communities comprising 20 bacterial strains mixed at varied proportions. The specificity, sensitivity, and quantification of our assay was also evaluated using clinical isolates and extracts from swine manure. We found that our single-tube, target-enriched library workflow accurately and reproducibly identified genomic content from reference strains with as few as six starting genomic copies mixed in complex samples. Further, enrichment of seven variable (V) regions of the 16S rRNA gene facilitated the detection of greater diversity and improved classification of microbial communities compared to the conventionally used primer set, which only targets the V3-V4 regions. Simultaneous analysis of both the 16S rRNA gene and antibiotic resistance genes provided greater differentiation of bacterial pathogens and greater characterization of the overall structure of the community’s functional repertoire. This study highlights the power of targeted enrichment via single-pool amplification for sequencing and characterizing microbial communities, including those that have important consequences for human health.

Citation Format: Kayla Peck, Robert Stedtfeld, Jordan RoseFigura, Brett Reed, Drew McUsic, Jon Irish, Brett Etchebarne, Timothy Johnson, Laurie Kurihara, Vladimir Makarov. Microbial sequencing using a single-pool target enrichment of multiple variable regions of the 16S rRNA gene, the nuclear ribosomal internal transcribed spacer (ITS) region, and antimicrobial resistance genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1484.