Abstract 1164: Targeted next generation sequencing microbiome assay design

Abstract

Abstract Background: Targeted Next Generation Sequencing may provide a lower cost approach for characterizing the Microbiome. Sequencing of 16S rRNA is one approach to characterize microbial diversity. However, 16S sequencing alone is often insufficient to gain species level information. Here we describe the design capabilities of targeted sequencing panels comprising both 16S and species-specific primers using Ion AmpliSeqTM technology. These types assay can be used to understand the composition and diversity of the microbiomes at the species level for human health and other applications. Method: Our assays include the capability to customize primers to multiple hypervariable regions of the 16S region. However, 16S alone can still be insufficient in distinguishing highly homologous organisms. To address this we can design species-specific primers that provide high strain coverage and high specificity performance. We have developed and tested example assays that include primers for a variety of species identification. With this approach, we can generate reads from sequencing to report taxonomic classifications and relative abundance for organisms in the sample with high specificity and sensitivity. Results: We designed many a multiplexed Ion AmpliSeqTM assays including 16S targets and species-specific markers targeting important microbial species that can be adapted to new species of interest. This approach is proven as example of our fixed panels including Ion AmpliSeq Microbiome Health research Panel, Pan Bacterial and Anti-Microbial Resistance (AMR) community panels using the Ion Genestudio S5™ system. With the data generated from the species-specific primers, we identified all the targeted species in the ATCC samples at the Species level. We also created mixed population pools using DNA from clonal isolates of different bacterial strains. We are able to identify most microbes at Genus level with 100% sensitivity using 16S alone. We improved the detection resolution to the Species level through the application of our species-specific primers. Conclusions: Using the Ion AmpliSeq™ technology, we can create highly sensitive and specific assays for robust characterization of microbiota customized to an individual labs needs. This highly multiplexed PCR approach enables an efficient and affordable means for conducting extensive analyses of the microbiomes having applications in the study of the etiology of phenotypic variability. For research use only. Not for use in diagnostic procedures. Citation Format: Aren Ewing. Targeted next generation sequencing microbiome assay design [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 1164.