Rolling Circle Reverse Transcription Enables High Fidelity Nanopore Sequencing of Small RNA

Small RNAs (sRNAs) are an important group of non-coding RNA that have great potential as diagnostic and prognostic biomarkers for the treatment of a wide variety of diseases. Nanopore based sequencers from Oxford Nanopore Technologies are ideal for point of care and low resource settings because of their ease of use, low equipment and consumable costs, small footprint and real-time data reporting capabilities; however currently small RNA cannot be reliably sequenced on these devices due to their short size and the relatively high error rate of the sequencer. We reasoned that rolling circle reverse transcription could be performed on circularized small RNA to produce long concatemers which could be sequenced on these devices. In this work we developed a highly efficient workflow to add adapters and circularize sRNAs and perform rolling circle reverse transcription which we call SR-Cat-Seq. Furthermore, we show that the highly processive Group II Intron reverse transcriptases are superior to retroviral reverse transcriptases in rolling circle reverse transcription. We show that our sequencing method can produce highly accurate sRNA consensus sequences and has similar performance to traditional short-read methods.