Nanopore-based native RNA sequencing provides insights into prokaryotic transcription, operon structures, rRNA maturation and modifications

Felix Grünberger; Robert Knüppel; Michael Jüttner; Martin Fenk; Andreas Borst; Robert Reichelt; Winfried Hausner; Jörg Soppa; Sebastien Ferreira-Cerca; Dina Grohmann

highly accurate yet short sequencing reads (commonly 100-300 bp). Hence, sequence information 62 is only available in a fragmented form, making full-length transcript-or isoform-detection a 63 challenging task 9,10 . Sequencing platforms developed by Pacific Bioscience (PacBio) and Oxford 64 Nanopore Technologies (ONT) solved this issue. Both sequencing methods are bona fide single-65 molecule sequencing techniques that allow sequencing of long DNAs or RNAs 11,12 . However, the 66 base detection differs significantly between the two methods. PacBio-sequencers rely on 67 fluorescence-based single-molecule detection that identifies bases based on the unique fluorescent 68 signal of each nucleotide during DNA synthesis by a dedicated polymerase 11 . In contrast, in an ONT 69 sequencer, the DNA or RNA molecule is pushed through a membrane-bound biological pore with 70 the aid of a motor protein that is attached to the pore protein called a nanopore (Fig. 1a) . A change 71 in current is caused by the translocation of the DNA or RNA strand through this nanopore, which 72 serves as a readout signal for the sequencing process. Due to the length of the nanopore (version 73 R9.4), a stretch of approximately five bases contributes to the current signal. Notably, only ONT 74 transcriptomes 19,21-23 and to detect RNA isoforms in eukaryotes 24, 25 . However, prokaryotic 82 transcriptomes have not been characterized on the genome-wide level by native RNA-seq 83 approaches so far as prokaryotic RNAs lack a poly(A) tail which is required to capture the RNA and 84 feed it into the nanopore. 85