Fig 1: Overview of library preparation workflows used in this study. a In the ONT-NSK007 cDNA library preparation method, polyA RNA is used as a template for first strand cDNA synthesis which is initiated from an oligodT primer. The NEB second strand cDNA synthesis module (E6111) is then used to generate double-stranded cDNAs; here random primers are used to initiate cDNA synthesis, the products of which are then stitched together by DNA ligase. Note that since priming of second strand synthesis occurs randomly, this may not always begin from the very end of the first strand template, as depicted in the example here. Adaptor–motor complexes are then ligated to the double-stranded cDNA ends, although in instances where the first strand overhang might be particularly long, as in the example here, it is unlikely that the adaptor–motor complex will ligate efficiently to enable sequencing of the second strand. b To better enrich for full-length cDNAs, the ONT-DCS108 direct cDNA sequencing kit, which leverages template-switching17, was used. When the first strand cDNA synthesis reaches the end of the RNA molecule, the reverse transcriptase will add a few non-templated Cs to the end of the cDNA. A strand-switching primer (SSP) present in the reaction binds to these non-templated Cs, and the reverse transcriptase then switches template from the RNA to the SSP. The second cDNA strand, presuming its synthesis continues to the end of the first strand template as in the example here, will also span the full length of the primary polyA RNA template; note, however, that in many instances a non-full-length second cDNA strand will likely be sequenced. c The ONT-RNA001 workflow enables sequencing of native RNA strands. Here an oligodT-adaptor-motor complex is ligated to the polyA end of the RNA. In order to relax the secondary structure of the RNA (and thus help ensure efficient translocation of the RNA strand through the nanopore), a cDNA synthesis step is performed. Since only the RNA strand has a motor ligated, the RNA molecule, but not the cDNA strand, is sequenced