Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

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

Abstract

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.

Authors:
; ; ; ; ; ORCiD logo
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:
1645170
Alternate Identifier(s):
OSTI ID: 1487413; OSTI ID: 1559518
Report Number(s):
SAND-2018-10906J; SAND2019-9606J
Journal ID: ISSN 2045-2322; 11475; PII: 47857
Grant/Contract Number:  
AC04-94AL85000
Resource Type:
Published Article
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Name: Scientific Reports Journal Volume: 9 Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United Kingdom
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 97 MATHEMATICS AND COMPUTING

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 Kingdom: N. p., 2019. Web. doi:10.1038/s41598-019-47857-3.
Copy to clipboard
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 Kingdom. https://doi.org/10.1038/s41598-019-47857-3
Copy to clipboard
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 Kingdom. https://doi.org/10.1038/s41598-019-47857-3.
Copy to clipboard
@article{osti_1645170,
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 = {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.},
doi = {10.1038/s41598-019-47857-3},
journal = {Scientific Reports},
number = 1,
volume = 9,
place = {United Kingdom},
year = {Wed Aug 07 00:00:00 EDT 2019},
month = {Wed Aug 07 00:00:00 EDT 2019}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1038/s41598-019-47857-3
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Citation Metrics:
Cited by: 12 works
Citation information provided by
Web of Science

Figures / Tables:

Figure 1 Figure 1: Schematic of the RUBRIC workflow illustrating the division of computational effort between two garden-variety PCs: a laptop that runs the MinION sequencer and its MinKNOW software interfaced through the Read Until API (via ethernet) to a desktop system that performs the key RUBRIC operations of pre-screening reads formore » admission to the decision process, basecalling and aligning reads to nucleic acid target reference(s) in real-time, and communicating any resulting skip/reject decisions back to MinKNOW.« less
All figures and tables (24 total)
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

Nanopore sequencing and assembly of a human genome with ultra-long reads
journal, January 2018

  • Jain, Miten; Koren, Sergey; Miga, Karen H.
  • Nature Biotechnology, Vol. 36, Issue 4
  • DOI: 10.1038/nbt.4060

Adaptive seeds tame genomic sequence comparison
journal, January 2011

CRISPR–Cas9-targeted fragmentation and selective sequencing enable massively parallel microsatellite analysis
journal, February 2017

  • Shin, GiWon; Grimes, Susan M.; Lee, HoJoon
  • Nature Communications, Vol. 8, Issue 1
  • DOI: 10.1038/ncomms14291

Metagenomics: The Next Culture-Independent Game Changer
journal, July 2017

  • Forbes, Jessica D.; Knox, Natalie C.; Ronholm, Jennifer
  • Frontiers in Microbiology, Vol. 8
  • DOI: 10.3389/fmicb.2017.01069

Evolution of selective-sequencing approaches for virus discovery and virome analysis
journal, July 2017

Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis
journal, September 2015

  • Greninger, Alexander L.; Naccache, Samia N.; Federman, Scot
  • Genome Medicine, Vol. 7, Issue 1
  • DOI: 10.1186/s13073-015-0220-9

In Situ Field Sequencing and Life Detection in Remote (79°26′N) Canadian High Arctic Permafrost Ice Wedge Microbial Communities
journal, December 2017

The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community
journal, November 2016

Use of the Oxford Nanopore MinION sequencer for MLST genotyping of vancomycin-resistant enterococci
journal, July 2017

Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella
journal, May 2015

Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool
journal, February 2016

  • Hoenen, Thomas; Groseth, Allison; Rosenke, Kyle
  • Emerging Infectious Diseases, Vol. 22, Issue 2
  • DOI: 10.3201/eid2202.151796

Nanopore sequencing detects structural variants in cancer
journal, January 2016

Highly parallel direct RNA sequencing on an array of nanopores
journal, January 2018

  • Garalde, Daniel R.; Snell, Elizabeth A.; Jachimowicz, Daniel
  • Nature Methods, Vol. 15, Issue 3
  • DOI: 10.1038/nmeth.4577

Real-time, portable genome sequencing for Ebola surveillance
journal, February 2016

  • Quick, Joshua; Loman, Nicholas J.; Duraffour, Sophie
  • Nature, Vol. 530, Issue 7589
  • DOI: 10.1038/nature16996

Forensic SNP Genotyping using Nanopore MinION Sequencing
journal, February 2017

  • Cornelis, Senne; Gansemans, Yannick; Deleye, Lieselot
  • Scientific Reports, Vol. 7, Issue 1
  • DOI: 10.1038/srep41759

Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing
journal, September 2016

  • Schmidt, K.; Mwaigwisya, S.; Crossman, L. C.
  • Journal of Antimicrobial Chemotherapy, Vol. 72, Issue 1
  • DOI: 10.1093/jac/dkw397

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome
journal, December 2017

  • Tyson, John R.; O'Neil, Nigel J.; Jain, Miten
  • Genome Research, Vol. 28, Issue 2
  • DOI: 10.1101/gr.221184.117

Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer
journal, June 2017

  • Suzuki, Ayako; Suzuki, Mizuto; Mizushima-Sugano, Junko
  • DNA Research, Vol. 24, Issue 6
  • DOI: 10.1093/dnares/dsx027

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples
journal, May 2017

  • Quick, Joshua; Grubaugh, Nathan D.; Pullan, Steven T.
  • Nature Protocols, Vol. 12, Issue 6
  • DOI: 10.1038/nprot.2017.066

A framework and an algorithm to detect low-abundance DNA by a handy sequencer and a palm-sized computer
journal, July 2018

High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell
journal, February 2018

Comparative analysis of targeted long read sequencing approaches for characterization of a plant’s immune receptor repertoire
journal, July 2017

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach
journal, February 2017

Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples
journal, March 2017

  • Votintseva, Antonina A.; Bradley, Phelim; Pankhurst, Louise
  • Journal of Clinical Microbiology, Vol. 55, Issue 5
  • DOI: 10.1128/jcm.02483-16

A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits
journal, August 2015

  • Karamitros, Timokratis; Magiorkinis, Gkikas
  • Nucleic Acids Research, Vol. 43, Issue 22
  • DOI: 10.1093/nar/gkv773

Single-molecule sorting of DNA helicases
journal, October 2016

MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island
journal, December 2014

  • Ashton, Philip M.; Nair, Satheesh; Dallman, Tim
  • Nature Biotechnology, Vol. 33, Issue 3
  • DOI: 10.1038/nbt.3103

Streaming algorithms for identification of pathogens and antibiotic resistance potential from real-time MinIONTM sequencing
journal, July 2016

Real-time selective sequencing using nanopore technology
journal, July 2016

  • Loose, Matthew; Malla, Sunir; Stout, Michael
  • Nature Methods, Vol. 13, Issue 9
  • DOI: 10.1038/nmeth.3930

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer
journal, May 2018

Design of targeted, capture-based, next generation sequencing tests for precision cancer therapy
journal, December 2013

Unbiased Strain-Typing of Arbovirus Directly from Mosquitoes Using Nanopore Sequencing: A Field-forward Biosurveillance Protocol
journal, April 2018

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel
journal, May 2012

  • Cipriany, B. R.; Murphy, P. J.; Hagarman, J. A.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 22
  • DOI: 10.1073/pnas.1117549109

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics
journal, November 2017

  • Brinkmann, Annika; Ergünay, Koray; Radonić, Aleksandar
  • PLOS Neglected Tropical Diseases, Vol. 11, Issue 11
  • DOI: 10.1371/journal.pntd.0006075

Culture-independent approaches to chlamydial genomics
journal, February 2018

  • Taylor-Brown, Alyce; Madden, Danielle; Polkinghorne, Adam
  • Microbial Genomics, Vol. 4, Issue 2
  • DOI: 10.1099/mgen.0.000145

Systematic and stochastic influences on the performance of the MinION nanopore sequencer across a range of nucleotide bias
journal, February 2018

    Sort by:
    [ × clear filter / sort ]

    Figures / Tables found in this record:

    Figure 1 (p. 2)figure
    Table 1 (p. 3)table
    Figure 2 (p. 4)figure
    Table 2 (p. 6)table
    Figure 3 (p. 7)figure
    Figure 4 (p. 8)figure
    Figure S1 (p. 14)figure
    Figure S2 (p. 15)figure
    Figure S3 (p. 15)figure
    Figure S4 (p. 16)figure
    Figure S5 (p. 17)figure
    Figure S6 (p. 18)figure
    Figure S7 (p. 19)figure
    Table S1 (p. 21)table
    Figure S8 (p. 22)figure
    Figure S9 (a-b) (p. 25)figure
    Figure S9 (c-d) (p. 26)figure
    Figure S9 (e-f) (p. 27)figure
    Figure S9 (g-h) (p. 28)figure
    Figure S9 (i-j) (p. 29)figure
    Figure S9 (k-l) (p. 30)figure
    Figure S9 (m-n) (p. 31)figure
    Figure S10 (a-h) (p. 32)figure
    Figure S10 (i-p) (p. 33)figure
      [ × clear filter / sort ]
      Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.

      Similar Records in DOE PAGES and OSTI.GOV collections: