All raw voltage traces were collected as FAST5 files using Oxford Nanopore Technologies’ software MinKNOW (versions 4.2.11 and 3.4.9). In order to minimize variability in basecalling and downstream analyses based on software versions, all libraries were reprocessed from FAST5s with the same pipeline as follows. Raw FAST5 files from MinKNOW were basecalled with Guppy (v6.0.1) in GPU mode using parameters: guppy_basecaller -c rna_r9.4.1_70bps_hac.cfg. Basecalled reads were aligned to C. elegans genome (WBCel235) using MiniMap2 (v2.17-r941) [13 (link)] with recommended settings for dRNA-seq: minimap2 -x splice -uf -k14. Additionally, parameter –junc-bed was used with a bed genome annotation file to provide minimap2 with splice junction information.
For libraries from Roach et al., 2020 [10 (link)], available FAST5 files were collected from the European Nucleotide Archive (ENA accession: PRJEB31791). All downstream processing of FAST5 files was identical to other processed libraries.
For libraries from Roach et al., 2020 [10 (link)], available FAST5 files were collected from the European Nucleotide Archive (ENA accession: PRJEB31791). All downstream processing of FAST5 files was identical to other processed libraries.
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