Revolutionizing Bacterial Diagnostics: A Genomic Analysis of Nanopore Sequencing

Abstract

Time is a crucial factor in making an appropriate diagnosis when a patient is seriously ill with a bacterial infection, especially with the looming threat of Antimicrobial Resistance. Traditional culture-based methods can take several days to even weeks to come back to the clinical team, with the patient usually treated with a course of broad-spectrum antibiotics for a bacterial infection in the meantime. Faster next-gen short-read sequencing platforms offer a rapid turnaround time providing bacterial species identification, but are unable to reliably sequence plasmids which can offer valuable information of what virulence factors or antimicrobial resistance genes might be present. Use of long-read sequencing platforms from Oxford Nanopore Technology and Pacific Biosystems are on the rise due to their ease of use, fast turnaround times, and cheap cost—but are almost always used in combination with short-read Illumina sequencing as a gold-standard to fill in the gaps where long-read sequencing falls short. Here, we examine Oxford Nanopore Technology’s MinION nanopore sequencing platform and its reliability to be used as a standalone method. For context, our work will be completed within the Brigham & Women’s Clinical Microbiology laboratory. Our main research question involves whether Oxford Nanopore Technology’s assays are reliable enough to be used for complete bacterial genome and extrachromosomal plasmid sequencing as a standalone method without the need for concurrent hybrid assembly. If a nanopore sequencing diagnostic assay is deemed reliable to be used as a standalone method, then it has further applications in a clinical setting for whole-genome as well as plasmid sequencing of bacterial pathogens—providing tailored genomic information in real time, allowing for better patient outcome. We plan on testing our hypothesis through use of Oxford Nanopore Technology (ONT)’s MinION nanopore sequencing technology and a hybrid assembly using both Illumina and Nanopore sequences with a bioinformatic pipeline developed in-house. Bacterial isolates obtained from cerebrospinal fluid (CSF) and blood cultures are the initial specimen source for this assay. Although there were variations observed from nanopore-only compared to hybrid sequencing, we concluded that Nanopore sequencing is reliable enough to be used on its own for species identification, but not for plasmid identification. This novel research is important as there have been no similar comparison studies to date. With an increased trust of Nanopore sequencing solo-use, providers will be armed with valuable genomic information to treat their patient in real time. This will greatly lessen the patient’s time on broad spectrum antibiotics, greatly improving antimicrobial stewardship.