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

Abstract

The Oxford MinION, the first commercial nanopore sequencer, is also the first to implement molecule-bymolecule 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.

Authors:
 [1];  [2];  [2];  [2];  [2]; ORCiD logo [2]
  1. (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States); Univ. of Toronto, ON (Canada)
  2. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Publication Date:
Research Org.:
Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1559518
Report Number(s):
SAND2019-9606J
Journal ID: ISSN 2045-2322; 678534
Grant/Contract Number:  
AC04-94AL85000
Resource Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Edwards, Harrison S., Krishnakumar, Raga, Sinha, Anupama, Bird, Sara W., Patel, Kamlesh D., and Bartsch, Michael S. Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria. United States: N. p., 2019. Web. doi:10.1038/s41598-019-47857-3.
Edwards, Harrison S., Krishnakumar, Raga, Sinha, Anupama, Bird, Sara W., Patel, Kamlesh D., & Bartsch, Michael S. Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria. United States. doi:10.1038/s41598-019-47857-3.
Edwards, Harrison S., Krishnakumar, Raga, Sinha, Anupama, Bird, Sara W., Patel, Kamlesh D., and Bartsch, Michael S. Wed . "Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria". United States. doi:10.1038/s41598-019-47857-3. https://www.osti.gov/servlets/purl/1559518.
@article{osti_1559518,
title = {Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria},
author = {Edwards, Harrison S. and Krishnakumar, Raga and Sinha, Anupama and Bird, Sara W. and Patel, Kamlesh D. and Bartsch, Michael S.},
abstractNote = {The Oxford MinION, the first commercial nanopore sequencer, is also the first to implement molecule-bymolecule 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.},
doi = {10.1038/s41598-019-47857-3},
journal = {Scientific Reports},
number = 1,
volume = 9,
place = {United States},
year = {2019},
month = {8}
}

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