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

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

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteriophage lambda / genetics
  • CRISPR-Cas Systems / genetics
  • DNA, Bacterial / genetics
  • DNA, Viral / genetics
  • Escherichia coli / genetics
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing / instrumentation
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Models, Genetic*
  • Nanopores
  • Proof of Concept Study
  • Sequence Analysis, DNA / instrumentation
  • Sequence Analysis, DNA / methods*

Substances

  • DNA, Bacterial
  • DNA, Viral