Characterization Of Gut Microbiota And Identification Of Key Bacterial Species Associated With Immune Response Using A New Ion Torrent Next-Generation Sequencing Assay

The gut microbiome affects immunity both locally and systemically; recent studies support a link between bacterial signatures in the gut and response to immune checkpoint blockade in cancer. Specifically, the gut microbiota is a potential biomarker to assess the effect of immune-modulatory drugs in the CTLA-4 and PD-1 pathways. A low cost targeted solution to characterizing and profiling microbial diversity in the gut is sequencing of the 16S rRNA gene. However, 16S sequencing alone is often insufficient to gain species level resolution, due to high homology across different bacteria. Here we describe a first-of-its-kind targeted sequencing solution based on Ion AmpliSeqTM technology that supplements 16S gene targets, with highly species-specific primers for a cohort of bacteria associated with cancer, response to cancer immunotherapy, and several gastrointestinal and auto-immune disorders. This assay can be used to better understand the composition and diversity of the human gut microbiome in the context of these phenotypes, as compared to other currently existing solutions. Our species-specific content provides great strain coverage and very high specificity performance for 73 different bacterial species including H. pylori, B. vulgatus and B. adolescentis, and several others from highly homologous genera like Lactobacillus and Bacteroides which are indistinguishable using just 16S targets. In addition, we have developed an approach to identify species-specific signatures/targets that easily extends to any new bacterial species that might become relevant in the future. Furthermore, our new end-to-end solution includes algorithms to automatically analyze reads from sequencing and generate a report with accurate taxonomic classifications, sample diversity metrics and relative abundance visualizations for organisms across samples. Using the Ion GeneStudio S5™ system, we sequenced two ATCC community controls comprising of 20 and 12 bacterial strains, and several custom mixed population samples containing DNA from 73 different bacterial species. With our more comprehensive 16S design covering 8 hyper-variable regions, we positively identified all the organisms at the genus level with 100% sensitivity. Using our species-specific primers, we identified all the targeted species with 100% sensitivity and specificity in both, the community controls and mixed population samples at the species level. We have created a highly sensitive and specific assay for robust characterization of gut microbiota. This highly multiplexed approach enables an efficient and affordable means for conducting extensive analyses of the human microbiome having applications in the study of phenotypic variability, and the potential relationship to disease, resistance, and therapy. For research use only. Not for use in diagnostic procedures. Citation Format: Shrutii Sarda, David C. Merrill, Heesun Shin, Anna McGeachy, Birgit Drews, Wing Lee, Rajesh Kumar Gottimukkala, Janice Au-Young, Fiona M. Hyland. Characterization of gut microbiota and identification of key bacterial species associated with immune response using a new Ion Torrent next-generation sequencing assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3347.