Comparison of Illumina and Oxford Nanopore Sequencing Technologies for Pathogen Detection from Clinical Matrices...

Next-generation sequencing is rapidly finding footholds in numerous microbiological fields, including infectious disease diagnostics. Here, we describe a molecular inversion probe panel for the identification of bacterial, viral, and parasitic pathogens. We describe the ability of Illumina and Oxford Nanopore Technologies (ONT) to sequence small amplicons originating from this panel for the identification of pathogens in complex matrices. The panel correctly classified 31 bacterial pathogens directly from positive blood culture bottles with a genus-level concordance of 96.7% and 90.3% on the Illumina and ONT platforms, respectively. Both sequencing platforms detected 18 viral and parasitic organisms directly from mock clinical samples of plasma and whole blood at concentrations of 10⁴ PFU/mL with few exceptions. In general, Illumina sequencing exhibited greater read counts with lower percent mapped reads; however, this resulted in no effect on limits of detection compared with ONT sequencing. Mock clinical evaluation of the probe panel on the Illumina and ONT platforms resulted in positive predictive values of 0.91 and 0.88 and negative predictive values of 1 and 1 from de-identified human chikungunya virus samples compared with gold standard quantitative RT-PCR. Overall, these data show that molecular inversion probes are an adaptable technology capable of pathogen detection from complex sample matrices on current next-generation sequencing platforms.