PRJNA491460
Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall- and Reference-Informed Criteria
The Oxford MinION, the first commercial nanopore sequencer, is also the first to implement molecule-by-molecule real-time selective sequencing or “Read Until”. As DNA transits a MinION nanopore, real-time pore current data can be accessed and analyzed to provide active feedback to that pore. DNA fragments of interest are sequenced by default, while DNA deemed non-informative is rejected by reversing the pore bias to eject the fragment, providing a novel means of target enrichment and/or background depletion. In contrast to the previously published pattern-matching Read Until approach, our RUBRIC method is the first example of real-time selective sequencing where on-line basecalling and alignment against conventional genomic (ACTG) references provide the basis for sequence/reject decisions. We evaluate RUBRIC performance across a range of optimizable parameters, apply it to mixed human/bacteria and CRISPR/Cas9-cut samples, and present a generalized model for estimating real-time selection performance as a function of sample composition and computing configuration.