Fig. 1: Real-time and programmable transcriptome sequencing with PROFIT-seq.
From: Real-time and programmable transcriptome sequencing with PROFIT-seq
a, A schematic overview of the PROFIT-seq method. First, the ribosomal RNA-depleted total RNA was reverse transcribed using a combinatorial RT strategy. Double-stranded oligo(dT) (dsdT), dsN and ssN were successively added to capture polyadenylated, non-polyadenylated and circular RNAs. The reverse-transcribed cDNA was then circularized and amplified using the RCA assay. The nanopore sequencing library was constructed, and PROFIT-seq was used for real-time control of the sequencing process. The acquired chunk data were basecalled and demultiplexed according to the sequencing time, channel number and detected barcodes. The basecalled sequences were subsequently aligned to the reference genome. Finally, PROFIT-seq determined whether the sequencing process should be continued or rejected according to the user-provided sequencing configuration. b, The length of rejected (purple) and finished (green) reads for canonical DNA, cDNA and PROFIT-seq runs. All bulk fast5 runs were simulated for sequencing all reads or rejecting all reads for 1 h. c, The elapsed time for raw signal basecalling, sequence alignment and pore manipulation for each acquired chunk of data. d, PROFIT-seq provides three manipulation modes, including enrichment or depletion of reads aligned to target regions and the balancing mode for dynamic determination of enriching targets based on the accomplished coverage. e, The performance of enriching chromosomes 1, 2, 5, 11 and 12 and depleting other chromosomes (middle) or balancing coverage of all chromosomes (right). Source numerical data are available in Source data.
