A Comprehensive qPCR-Based Oral Microbiome Profiling Platform for Research and Discovery

The oral microbiome plays a crucial role in dental health and can offer insights into a wide variety of systemic conditions. Next-generation sequencing (NGS) has been used to study the oral microbiome, but 16S amplicon sequencing and shotgun metagenomic sequencing have severe limitations as tools for biomarker discovery and clinical diagnostics. Saliva is a readily available sample type that contains over a billion microbes per mL, but the signal from low abundance microbes and correct species identification can be difficult to detect without deep, expensive shotgun metagenomic sequencing. qPCR-based molecular detection can overcome the limitations of NGS methods by offering specificity, sensitivity, and quantification down to single digit copy numbers. Branchpoint Biosciences, an early-stage start-up, has developed a qPCR platform technology to create a new generation of qPCR assays. Our new method combines cutting-edge knowledge of genomic databases and microbial evolution within a proprietary software suite that enables the rapid and accurate design of discriminatory primers and probes capable of detecting target microorganisms without cross-reactivity. We have de-risked the underlying software suite, established in silico and in vitro validation methods, and successfully demonstrated a proof-of-concept panel consisting of 106 validated qPCR assays with high specificity and sensitivity of microbial targets in complex samples. In this Phase 1 proposal, we plan to develop a comprehensive oral qPCR panel through the following aims: (1) determine oral panel targets by ascertaining abundance and prevalent microbes in the oral microbiome from 926 public metagenomic samples studying six different disease phenotypes; and (2) design and validate qPCR assays targeting the identified microbes. A comprehensive qPCR panel of oral species creates a cost-effective research tool that does not require expensive pre- and post-processing steps associated with NGS techniques and can quantify all the Bacteria and Archaea commonly present in the human oral cavity. This panel will provide researchers and clinicians with an alternative to NGS techniques and enable large-scale cohort and/or detailed longitudinal studies for monitoring disease progression and therapeutic response. Successful completion of this Phase 1 proposal will result in the (1) identification of microbial targets and (2) demonstrate validated qPCR assays that accurately screen the oral microbiome. This work will precede a Phase 2 proposal that quantifies the advantages of generating a microbiome profile with our oral comprehensive panel compared to NGS, such as lower cost, faster turnaround time, superior detection, and quantitative values. Our novel platform technology enables a new, comprehensive, and cost-effective approach for discovering and leveraging microbial biomarkers for clinical research and sets the stage for the future development of microbiome-based monitoring platforms, screening tests, and diagnostics.