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Sci Rep. 2019 Aug 7;9(1):11475. doi: 10.1038/s41598-019-47857-3.

Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria.

Author information

1
Exploratory Systems Dept., Sandia National Laboratories, Livermore, CA, USA.
2
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada.
3
Systems Biology Dept., Sandia National Laboratories, Livermore, CA, USA.
4
Biotechnology & Bioengineering Dept., Sandia National Laboratories, Livermore, CA, USA.
5
uBiome, San Francisco, CA, USA.
6
Purdue Partnerships Dept., Sandia National Laboratories, Albuquerque, NM, USA.
7
Exploratory Systems Dept., Sandia National Laboratories, Livermore, CA, USA. mbarts@sandia.gov.

Abstract

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 background depletion and/or target enrichment. 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.

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