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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. Fragments of interest are sequenced by default, while DNA deemed non-informative is rejected by reversing the pore bias to eject the strand, 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 enables alignment against conventional nucleic acid references to 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.

RUBRIC is available via Github: https://github.com/harrisonedwards/RUBRIC

Authors: Harrison S Edwards, Raga Krishnakumar, Anupama Sinha, Sara W Bird, Kamlesh D Patel, Michael S Bartsch

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