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

An advantage of the long-read Nanopore sequencing technique is that native RNA strands can be 365 sequenced directly as near full-length transcripts 55 . This is also reflected in the sequenced data 366 sets as aligned lengths up to 7864 nt can be observed (see H. volcanii NOTEX sample with a median 367 read length of 1390 nt, Supplementary Fig. 3c ). The majority of reads from E. coli and H. volcanii 368 samples mapped to the 23S rRNA (Fig. 1b , see Supplementary Fig. 3a ). In general, the read identity 369 of CDS-mapping reads is higher than for rRNA mapping reads, but lower than the spike-in control 370 (see Supplementary Fig. 3b,c) . It is noteworthy, that a minimum aligned length of about 100 nt 371 appears in the data sets (see Supplementary Fig. 3d ) when using the native RNA sequencing 372 protocol and minimap2 as mapping tool 16,40,56 . Unaligned reads had a median read length of 191 373 nt, in contrast to 572 nt for aligned reads (all data sets combined) suggesting that short reads could 374 not be aligned properly. As small RNAs, CRIPSR-RNAs or tRNAs fall below this threshold, we 375 the author/funder. All rights reserved. No reuse allowed without permission.