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A growing number of methods are being developed and shared by the scientific community around the world for use in COVID-19-related work. A selection of preprints and protocols is shown below. These include methods for sequencing of SARS-CoV-2 and metatranscriptome analysis of COVID-19 samples, with applications in areas including epidemiology, metagenomic analysis and diagnostics.

Please note that these are preprints, protocols, and early access publications, and have not completed a peer-review process.

Nanopore sequencing

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nCoV-2019 sequencing protocol v2
Quick, J.
University of Birmingham, ARTIC Network

ARTIC amplicon sequencing protocol for MinION for nCoV-2019, incorporating one-pot native barcoding.

  • Q5® Hot Start High-Fidelity DNA Polymerase (NEB #M0493)
  • NEBNext® Ultra™ II End Repair / dA-Tailing Module (NEB #E7546)
  • NEBNext Ultra II Ligation Module (NEB #E7595)
  • NEBNext Quick Ligation Module (NEB #E6056)

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PCR tiling of COVID-19 virus
Oxford Nanopore Technologies

Based on the ARTIC Network protocol by Josh Quick (see above), this method generates 400bp tiled amplicons, followed by Oxford Nanopore Technologies sequencing.

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Long reads nanopore sequencing to recover SARS-CoV-2 whole genome  v.3
Resende, P.
Oswaldo Cruz Institute; Pathogen Genomics Unit and UCL Genomics, University College London

This protocol builds long (~2kb) amplicon libraries of SARS-CoV-2 genomes, suitable for nanopore sequencing.

  • Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494)
  • NEBNext Ultra II End Repair / dA-Tailing Module (NEB #E7546)
  • NEBNext Ultra II Ligation Module (NEB #E7595)
  • NEBNext Quick Ligation Module (NEB #E6056)

Illumina sequencing

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COVID-19 ARTIC v3 Illumina library construction and sequencing protocol
DNA Pipelines R&D et al.
Wellcome Sanger Institute

Using the ARTIC SARS-CoV-2 primers, this protocol generates libraries of 400bp tiled amplicons, followed by sample pooling and sequencing on the Illumina NovaSeq.

  • LunaScript RT SuperMix Kit (NEB #E3010)
  • Q5® Hot Start High-Fidelity 2X Master Mix (NEB #M0494)
  • NEBNext Ultra II DNA Library prep kit for Illumina (NEB #E7645)

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SARS-CoV-2 Enrichment Sequencing by Spiked Primer MSSPE method V.4
Manning, J.E. et al.
National Institute for Allergies and Infectious Diseases, Chan Zuckerberg Biohub, Institute Pasteur Cambodge

Incorporating a spiked primer enrichment step, this protocol generates libraries suitable for sequencing on the Illumna iSeq100.

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Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions
Butler, D.J. et al.
Weill Cornell Medicine, New York Genome Center, Baylor College of Medicine, HudsonAlpha Discovery Institute, University Hospital Tuebingen, Columbia University, New England Biolabs

This preprint describes a large-scale shotgun metatranscriptomic profiling platform for nasopharyngeal swabs, with Illumina sequencing, and use of Colorimetric LAMP to detect SARS-CoV-2 infection.

  • WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB #M1800)
  • NEBNext rRNA depletion v2 (Human/Mouse/Rat) (NEB #E7400)
  • NEBNext Ultra II Directional RNA (NEB #E7760)
  • NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (NEB #E6440)

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Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with 2019 Novel Coronavirus Disease, United States
Harcourt, J. et al.
Centers for Disease Control and Prevention, Eagle Medical Services, Oak Ridge Institute for Science and Education, Synergy America, Inc., University of Texas Medical Branch, World Reference Center for Emerging Viruses and Arboviruses

This publication includes RNA library preparation from passaged virus sequencing on the Illumina MiniSeq.

  • NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770)

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Early Spread of SARS-Cov-2 in the Icelandic Population
Gudbjartsson, D.F. et al.
deCODE genetics, University of Iceland, Landspitali University Hospital, Directorate of Health, Reykjavik

Using the ARTIC tiled primer method, the authors generated libraries which were pooled and sequenced on the Illumina MiSeq.

  • NEBNext Ultra II DNA Library Prep Kit for Illumina
  • Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494)

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LAMP-Seq: Population-Scale COVID-19 Diagnostics Using a Compressed Barcode Space
Schmid-Burgk, J.L. et al.
Broad Institute of MIT and Harvard, Massachusetts Institute of Technology, University Hospital Bonn, University of Washington, Dana-Farber Cancer Institute, German Cancer Research Center (DKFZ), National Center for Tumor Diseases (NCT), Heidelberg, Howard Hughes Medical Institute

This method proposes use of barcoded RT-LAMP followed by large-scale pooling, PCR amplification with additional barcoding and deep sequencing on the Illumina platform.

  • WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB #M1800)
  • Bst 3.0 DNA Polymerase (NEB #M0374)
  • NEBNext High-Fidelity 2X PCR Master Mix (NEB #M0541)



One or more of these products are covered by patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please email us at [email protected]. The use of these products may require you to obtain additional third party intellectual property rights for certain applications.

 

 

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