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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-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. As a result, 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];  [1];  [1];  [1];  [2];  [3]
  1. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  2. uBiome, San Francisco, CA (United States)
  3. Univ. of Toronto, ON (Canada)
Publication Date:
Research Org.:
Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1487413
Report Number(s):
SAND-2018-10906J
668444
Grant/Contract Number:  
AC04-94AL85000
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Methods / BioRxiv
Additional Journal Information:
Journal Name: Nature Methods / BioRxiv
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 97 MATHEMATICS AND COMPUTING

Citation Formats

Bartsch, Michael S., Krishnakumar, Raga, Sinha, Anupama, Patel, Kamlesh D., Bird, Sara W., and Edwards, Harrison S. Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria. United States: N. p., 2018. Web. doi:10.1101/460014.
Bartsch, Michael S., Krishnakumar, Raga, Sinha, Anupama, Patel, Kamlesh D., Bird, Sara W., & Edwards, Harrison S. Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria. United States. doi:10.1101/460014.
Bartsch, Michael S., Krishnakumar, Raga, Sinha, Anupama, Patel, Kamlesh D., Bird, Sara W., and Edwards, Harrison S. Fri . "Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria". United States. doi:10.1101/460014. https://www.osti.gov/servlets/purl/1487413.
@article{osti_1487413,
title = {Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria},
author = {Bartsch, Michael S. and Krishnakumar, Raga and Sinha, Anupama and Patel, Kamlesh D. and Bird, Sara W. and Edwards, Harrison S.},
abstractNote = {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. As a result, 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 = {10.1101/460014},
journal = {Nature Methods / BioRxiv},
number = ,
volume = ,
place = {United States},
year = {2018},
month = {11}
}

Journal Article:
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