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

As some bioinformatic tools depend on single-read files we first converted multi-read FAST5 files 185 from the MinKNOW output to single-read FAST5 files using the ont_fast5_api from Oxford 186 Nanopore (https://github.com/nanoporetech/ont_fast5_api). To prevent actual good-quality 187 reads from being discarded (this issue was reported previously 13,37 ), we included both failed and 188 passed read folders in the following steps of the analysis. Demultiplexing was done by poreplex 189 (version 0.4, https://github.com/hyeshik/poreplex) with the arguments --trim-adapter, --190 symlink-fast5, --basecall and --barcoding, to trim off adapter sequences in output FASTQ files, 191 basecall using albacore, create symbolic links to FAST5 files and sort the reads according to their 192 barcodes. However, to ensure consistency between non-multiplexed and multiplexed samples and 193 because of some major improvements in the current basecalling software (guppy), albacore files 194