![]() ![]() Capture regions were amplified by using 0.5 μmol/L forward and reverse sequencer-specific universal primers ( Table 1) and 1× Phusion HF MM (Qiagen). Reactions were heated to 37☌ for 30 minutes followed by inactivation at 80☌ for 20 minutes. Noncircular DNA was removed with the addition of 20 U of exonuclease I (Epicentre) and 25 U of exonuclease III (Epicentre). Reactions were heated to 98☌ for 3 minutes and then ramped to 55☌ (0.1☌ per second) with a 60-minute hold at 55☌ followed by 72☌ for 15 minutes. Louis, MO), 1× Phusion High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs, Ipswich, MA), 100 pmol/L PIP probe pool, and 2 μL of target organism DNA. 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.īriefly, 10 μL reaction mixtures contained 10 U of Ampligase (Epicentre, Madison, WI), 500 μmol/L nicotinamide adenine dinucleotide (Sigma-Aldrich, St. 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. 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. Both sequencing platforms detected 18 viral and parasitic organisms directly from mock clinical samples of plasma and whole blood at concentrations of 10 4 PFU/mL with few exceptions. 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. ![]() 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. Here, we describe a molecular inversion probe panel for the identification of bacterial, viral, and parasitic pathogens. Next-generation sequencing is rapidly finding footholds in numerous microbiological fields, including infectious disease diagnostics. ![]()
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