Next-day whole genome sequencing and analysis using nanopore sequencing for real-time outbreak and transmission determination in healthcare facilities

The goal of this project is to develop an ultra-rapid, cost-effective whole genome sequencing based method to determine if a healthcare associated infection (HAI) transmission event has occurred. HAIs, particularly those caused by multidrug resistant organisms, are a recognized healthcare threat. Despite the potential of whole genome sequencing technologies, there are no commercial offerings that can reliably detect an HAI transmission event within 24 hours. Rapid turnaround times resulting in the confirmation of an HAI event promise to reduce the spread of the HAI and has the potential to minimize the overuse and misuse of antimicrobial drugs. Likewise, a technology with the sensitivity and precision to rule out an HAI event will reduce unnecessary and expensive operational costs. To meet this medical and commercial need, Day Zero Diagnostics is developing a whole genome sequencing and analysis workflow that leverages the speed and other advantages of third generation sequencing technology (Oxford Nanopore) to demonstrate the feasibility of a 24 hour turnaround HAI service. In addition to rapid read generation, the key features that differentiate this approach from existing pipelines (e.g. Illumina technology) are that it overcomes the need for an external closely related reference genome and enables full genome assembly including mobile genetic elements (plasmids) that are a source of antimicrobial resistance and can be disseminated independently of the chromosomal genome. The goal of this Phase I effort is to develop the necessary operational pipelines generating processing, assembly, and annotation of third generation sequencing data for HAI. Aims are focused on the development of a single nucleotide polymorphism (SNP) variant calling algorithm that is optimized for Oxford Nanopore data (Aim 1); developing a de novo assembly and annotation pipeline for both chromosomal mobile element genomes (Aim 2), and demonstrating preliminary utility as an HAI investigation service (Aim 3). The successful completion of the aims of this proposal will advance the workflow towards further optimization and refinement, resulting in a commercial product with a “same day,” 24- hour turnaround time. The final product will be offered as a service that has the potential for routine use in hospital transmission investigations, therefore improving the capabilities of infection control and the management of HAIs.Healthcare associated infections affect 4% of patients in the United States, and multidrug resistant pathogens can lead to deadly hospital outbreaks. Current methods for determining whether a transmission event has occurred in a healthcare facility are slow, expensive, and difficult to scale. This project aims to fill that need with an ultra-rapid, scalable, and cost-effective analysis pipeline that will provide reliable, same day, transmission information in a window of time that can have a significant impact on the cost and magnitude of interventions a healthcare facility may employ.