Sequencing and COVID-19

NEBNext^® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies^®)

This kit now includes two options for balanced primer pools (VarSkip Short v2 for improved variant coverage including with Omicron, and ARTIC V3) plus reagents optimized for RT-PCR from SARS-CoV-2 gRNA and downstream library preparation. The method increases uniformity of genome coverage, across a wide copy number range, and is based on the original work of the ARTIC Network.

nCoV-2019 sequencing protocol v3 (LoCost)
Quick, J.
University of Birmingham

This new version of the ARTIC protocol updates the RT and ligation reagents used, reduces reaction volumes and enables increased multiplexing. This updated method is also described in a bioRxiv preprint Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore.

• LunaScript RT SuperMix Kit (NEB #E3010)
• Q5^® Hot Start High-Fidelity 2X Master Mix (NEB #M0494) or Q5 Hot Start High-Fidelity DNA Polymerase (NEB #M0493)
• Deoxynucleotide (dNTP) Solution Mix (NEB #N0447)
• Nuclease-free Water (NEB #B1500S)
• NEBNext Ultra II End Repair / dA-Tailing Module (NEB #E7546)
• Blunt/TA Ligase Master Mix (NEB #M0367)
• NEBNext Quick Ligation Module (NEB #E6056)

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)

PCR tiling of SARS-CoV-2 virus with Native Barcoding Expansion 96 (EXP-NBD196)
Oxford Nanopore Technologies

This method, based on the ARTIC Network protocol by Josh Quick (see above), generates 400bp tiled amplicons and includes updated cDNA synthesis and ligation steps. The method enables additional barcoding and is compatible with Oxford Nanopore Technologies sequencing on MinION or GridION.

• LunaScript RT SuperMix Kit (NEB #E3010)
• Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494)
• Blunt/TA Ligase Master Mix (NEB #M0367)
• NEBNext Ultra II End Repair / dA-Tailing Module (NEB #E7546)
• NEBNext Quick Ligation Module (NEB #E6056)

PCR tiling of SARS-CoV-2 virus
Oxford Nanopore Technologies

This updated version of this protocol, based on the ARTIC Network protocol by Josh Quick, generates 400bp tiled amplicons, followed by Oxford Nanopore Technologies sequencing. This version includes updated cDNA synthesis and ligation steps.

• LunaScript RT SuperMix Kit (NEB #E3010)
• Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494)
• Blunt/TA Ligase Master Mix (NEB #M0367)
• NEBNext Ultra II End Repair / dA-Tailing Module (NEB #E7546)
• NEBNext Quick Ligation Module (NEB #E6056)

nCoV-2019 environmental sample sequencing protocol
Diaz-Munos, S. et al.
University of California, Davis
 
This modification of the nCoV-2019 sequencing protocol v2 (GunIt) V.2 enables ARTIC MinION sequencing from environmental samples, including low concentration samples.  

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

This fast, scalable method has testing and screening applications, employing LAMP-based amplification followed by library construction and nanopore sequencing. 

• WarmStart LAMP Kit (DNA & RNA) (NEB# E1700)

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)

Rapid detection of SARS-CoV-2 and other respiratory viruses by using LAMP method with Nanopore Flongle workflow
Li, J. et al.
GrandOmics Genomics, GrandOmics Diagnostics, Wuhan and Beijing, China.

This method combines LAMP with the Oxford Nanopore Flongle workflow, to detect SARS-CoV-2 and other respiratory viruses.

• Bst 2.0^® DNA Polymerase (NEB #M0537)

NEBNext^® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina^®)
NEBNext^® ARTIC SARS-CoV-2 Library Prep Kit (Illumina^®)
 
These kits include balanced primer pools plus reagents optimized for RT-PCR from SARS-CoV-2 gRNA and downstream library preparation, including enzymatic cDNA fragmentation. The E7658 kit now includes two options for balanced primer pools (VarSkip Short v2 for improved variant coverage including with Omicron, and ARTIC V3), and generates library inserts in the 150 bp range, compatible with 2 x 75 sequencing on Illumina instruments. The E7650 kit libraries are ~400 bp, compatible with 2 x 250 sequencing. RT reaction conditions are the same for all input amounts, and a novel DNA polymerase eliminates the need to normalize amplicon concentrations prior to library preparation. The methods increase uniformity of genome coverage, across a wide copy number range, and are based on the original work of the ARTIC Network.

SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses
Turner, J.S. et al.
Washington University School of Medicine, Icahn School of Medicine at Mount Sinai, The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs

This publication demonstrates the use of immune repertoire analysis of prior infection with SARS-CoV-2.

• NEBNext Immune Sequencing Kit (Human) (NEB #E6320)

The authors used whole genome SARS-CoV-2 sequencing, using the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) to surveil SARS-CoV-2 positive samples.

• NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) (NEB #E7658)

COVID-19 ARTIC v3 Illumina library construction and sequencing protocol V.5
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)

COVID-19 ARTIC v3 Illumina library construction and sequencing protocol - high throughput 384 format V.2
DNA Pipelines R&D et al.
Wellcome Sanger Institute

For higher density of sample processing, this adaptation of the COVID-19 ARTIC v3 protocol is compatible with 384 samples.

• 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)

COVID-19 ARTIC v3 Illumina library construction and sequencing protocol - short amplicons (275bp)
Betteridge, E. et al.
Wellcome Sanger Institute and University of Birmingham

This protocol describes production of 275nt amplicons, followed by library preparation and sample pooling. Any Illumina instrument with a 300 cycle (or greater) kit can then be used for sequencing.

• 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)

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

This adaptation of the COVID-19 ARTIC v3 protocol makes libraries directly from the 400bp tiled amplicons, using a second PCR step and without conventional library construction.

• LunaScript RT SuperMix Kit (NEB #E3010)
• Q5^® Hot Start High-Fidelity 2X Master Mix (NEB #M0494)

This tailed amplicon protocol generates 275bp tiled amplicons, compatible with 2 x 150 paired end sequencing.

• LunaScript RT SuperMix Kit (NEB #E3010)
• NEBNext Ultra™ II Q5^® Master Mix (NEB #M0544)
• Q5^® Hot Start High-Fidelity 2X Master Mix (NEB #M0494)

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.

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

This publication includes 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)
• NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB #E6111)
• Random Primer 6 (NEB #S1230)
• Protoscript II First Strand cDNA Synthesis Kit (NEB #E6560)

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)

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)

Introductions and early spread of SARS-CoV-2 in France
Gambaro, F. et al.
Institute Pasteur, Paris; National Influenza Centre, Viral Respiratory Laboratory, Algiers; Université de Paris, Paris

The authors sequenced samples collected in France between January and March, 2020, including samples with low viral loads (<10,000 viral copies/ul of extracted RNA).

• NEBNext Ultra II DNA Library prep kit for Illumina (NEB #E7645)
• NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (NEB #E6440)

LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding
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)