Setup of direct RNA-seq adaptive sampling (DRAS) using an in vitro transcripts (IVTs) model system. (A) Schematic representation of the AS principle. RNA strands ligated to adapters with motor protein are captured and sequencing is initiated. Small data chunks are sent for basecalling and alignment to a provided FASTA or BED file (left panel). If these data chunks are too small (<200 nt) for a reliable classification or mapping to multiple sites, more data chunks are collected until enough data for a decision are available (middle panel). In the enrichment case (vice versa for the depletion case), a reference match results in acceptance of the read, while no reference match results in rejection of the read (right panel). (B) Schematic representation of the classification of reads in AS. During the sequencing run, two files are generated that can be used to identify accepted and rejected reads, “adaptive sampling” and “sequencing summary.” The “adaptive sampling” file provides information on the decision that was made on individual sequencing chunks. Here, “stop_receiving” corresponds to accepted reads, whereas “unblock” represents rejected reads. Very short chunks cannot be classified and are categorized as “no_decision.” The “sequencing summary” file provides information on the end reason for every read, which is classified as: “signal_positive” (read passed pore completely), “data_service_unblock_mux_change” (read was rejected during adaptive sampling), “signal_negative” (current delta of 80 pA was observed), and “unblock_mux_change” (strand blocked pore and was rejected). (C–G) Equimolar mixtures of IVT1 (1869 nt) and IVT2 (1452 nt) were sequenced on Flongle flow cells in normal sequencing (NS) mode (C), AS with enrichment of IVT2 (D,E), or AS with depletion of IVT1 (F,G). The obtained reads were aligned to the reference sequences and split into rejected (D,F) and accepted reads (E,G) based on the sequencing summary.
