NCM 2022: Streamlining nanopore sequencing protocols for untargeted RNA virus detection
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Sequencing technology has improved in leaps and bounds in the recent past; however, RNA can still prove difficult and time consuming to sequence. Oftentimes, RNA extraction requires hazardous reagents, a multitude of consumables, and a lengthy cDNA conversion step. To make RNA virus sequencing more accessible, and potentially field-deployable, these obstacles need to be overcome. We evaluated numerous workflows with various kits, reagents, and consumables to find an optimal solution. In testing, we found that RNA-binding magnetic bead workflows could be employed for RNA extraction, with modifications to replace the more hazardous reagents from the waste-stream, reduce incubation times, and number of steps. Using magnetic-bead based extraction also eliminates the need for a microcentrifuge. The RNA yield and quality were sufficient for reverse transcription and final preparation of the cDNA with the Rapid Sequencing Kit. Total preparation time was under 90 minutes and it was possible to generate up to 913,440 total reads, 810,457 passing reads, and a Q Score of 12 after 20h sequencing with high accuracy base-calling. Centrifuge-or Minimap2-based algorithms were used to map reads and identify the organism. This decrease in time, removal of hazardous reagents, and use of Oxford Nanopore Technology sequencing allows for a more fieldable workflow to identify RNA viruses.
