B-239 Long-read Nanopore Sequencing is a Practicable Method of Targeted or Untargeted Microbial Contamination Detection

Abstract Background Sequencing-based methods for microbe detection are poised to replace both in vivo and PCR-based detection methods due to advantages in speed, sensitivity, specificity, and multiplexing. Here we describe pilot experiments to assess the practicability of nanopore sequencing for microbe detection. Methods Quantitative genomic Human Adenovirus 5 or Mycoplasma hominis DNA was spiked into remnant human serum and nucleic acids were extracted. 16S rRNA PCR amplification and barcoding was performed for Mycoplasma hominis identification. Sequencing was performed with a R9.4.1 flowcell on the MinION sequencer. Basecalling and genome alignment was performed with Guppy v.6.4.2. Results The performance of untargeted sequencing was evaluated by spiking quantitative Adenovirus DNA at 104 genome copies per microliter. Illustrative data from a single run is presented. Replicates yielded similar results. 1 520 991 reads between 31 bases and 970 kilobases were generated. 162 reads between 700 bases and 11.3 kilobases aligned to the Adenovirus genome (Image 1). To evaluate the performance of PCR amplified sequencing, we spiked Mycoplasma genomic DNA into serum or water. The entire 16S rRNA gene (1.5 kb) was PCR amplified and genomic DNA was spiked in at 104, 102, 10, and 0 (control) copies per microliter. A 10% signal: noise ratio cutoff yielded 100% specificity between spiked samples and negative controls. Conclusion Short read sequencing techniques are adequate for microbial detection but are capital intensive and prone to false positive results. We demonstrate that nanopore sequencing can generate long reads (>1.5 kb) with high-confidence microbe genome alignment. Our methods were sensitive to 104 and 10 copies per microliter utilizing untargeted and targeted methods, respectively. Further method optimization and validation will need to be performed, but these results demonstrate that nanopore sequencing is a practicable approach for microbe detection.