Product Class: Kit

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

The Small size of this product was discontinued on December 15, 2024. The Large size (NEB #E7660L) will continue to be available.

For improved performance with BA.2 variants, spike-in primers can now be provided, for use in combination with VarSkip Short v2 primers. Please see this FAQ for more information, including primer sequences. 

See below, including Figure 1 in the Product Information section, for information on VarSkip Short v2 performance with Omicron. Information on primer variant overlaps can be found here. 

A bulk format (2,304 reactions) is also available. Please contact us for more information.


Catalog #E7660

Product Introduction

The NEBNext® ARTIC kits were developed in response to the critical need for reliable and accurate methods for SARS-CoV-2 sequencing. These kits, for long and short read sequencing, were based on the original work of the ARTIC Network(1). The ARTIC SARS-CoV-2 sequencing workflow is a multiplexed amplicon-based whole-viral-genome sequencing approach. 

NEBNext ARTIC kits include primers and reagents for RT-PCR from SARS-CoV-2 gRNA and downstream library preparation for Illumina® and Oxford Nanopore Technologies® sequencing.

This kit includes two options for primers, including new VarSkip Short v2 primers:

  1. The new VarSkip (for Variant Skip) Short v2 primers have been designed at NEB to reduce the impact of SARS-CoV-2 variants on amplification and provide improved performance, including with the Omicron variant. The primer pools are balanced for coverage uniformity. Information on primer variant overlaps can be found here.

  2. The V3 ARTIC primers have been balanced, using methodology developed at NEB based on empirical data from sequencing. In combination with optimized reagents for RT-PCR, the kits deliver improved uniformity of amplicon yields from gRNA across a wide copy number range. 

The Express protocol option removes clean ups of cDNA amplicons and after ligation.

The NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) is compatible with sequencing on the Oxford Nanopore Technologies (ONT) platform and is used alongside kits from ONT to generate libraries with inserts in the 400 bp range, that can be sequenced on any ONT instrument, in 2-24 hour run times. 

If Illumina-compatible libraries are preferred, the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) (NEB #E7658) and the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) (NEB #E7650) generate libraries with ~150 bp and ~400 bp inserts, respectively. If only the reagents for cDNA synthesis and targeted cDNA amplification are required, the NEBNext ARTIC SARS-CoV-2 RT-PCR Module (NEB #E7626) is available. Note that E7650 does not include VarSkip v2 primers.

  • Includes two primer options: VarSkip Short v2, for improved Omicron coverage, or ARTIC V3
  • Information on primer variant overlaps can be found here
  • Improved uniformity of SARS-CoV-2 genome coverage, with balanced primer pools
  • Express protocol option available, with reduced cleanup steps
  • Streamlined, high-efficiency protocol
  • Effective with a wide range of viral genome inputs (10-10,000 copies)
  • For a bulk format of the kit (2,304 reactions) please contact us

1.(Josh Quick 2020. nCoV-2019 sequencing protocol v2 (GunIt). protocols.io https://dx.doi.org/10.17504/protocols.io.bdp7i5rn).

Product Information

Description

The ARTIC SARS-CoV-2 protocol from the ARTIC Network is a multiplexed amplicon approach covering the whole viral genome. The NEBNext ARTIC SARS-CoV-2 Library Prep Kit is based on this method and incorporates optimized and novel reagents. 

A single RT protocol is used regardless of input amount (10-10,000 viral genome copies), and no normalization step is required ahead of targeted amplification.

This kit includes two options for primers, including new VarSkip Short v2 primers:

  1. The VarSkip (for Variant Skip) Short primers have been designed at NEB to reduce the impact of SARS-CoV-2 variants on amplification and provide improved performance, including with the Omicron variant. VarSkip Short v2 primer pools generate ~550bp amplicons. Please see the FAQ section and the product manual for additional detail. Information on primer variant overlaps can be found here.

  2. The V3 ARTIC primers have been balanced, using methodology developed at NEB based on empirical data from sequencing. In combination with optimized reagents for RT-PCR, the kits deliver improved uniformity of amplicon yields from gRNA across a wide copy number range.

The Express protocol removes the cleanup of cDNA Amplicons.

The NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) is compatible with sequencing on the Oxford Nanopore Technologies platform and is used alongside kits from Oxford Nanopore Technologies to generate libraries with inserts in the  400bp range, that can be sequenced on any Oxford Nanopore Technologies instrument, in 2-24 hour run times.

If Illumina-compatible libraries are preferred, the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) (NEB #E7658) and the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) (NEB #E7650) generate libraries with ~150 bp and ~400 bp inserts, respectively. If only the reagents for cDNA synthesis and targeted cDNA amplification are required, the NEBNext ARTIC SARS-CoV-2 RT-PCR Module (NEB #E7626) is available.  Note that E7650 does not include VarSkip v2 primers.

Figure 1: Sequence coverage of a SARS-CoV-2 Omicron clinical sample with updated primer schemes



Caption: Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (log scale). Amplicons were generated from the same Omicron variant SARS-CoV-2 viral gRNA clinical sample using NEBNext® VarSkip Short SARS-CoV-2 primer pools, NEBNext VarSkip Short v2 SARS-CoV-2 primer pools, MilliporeSigma ARTICv4, MilliporeSigma ARTICv4 primer pools plus IDT ARTICv4.1 spike-in primers (ARTICv4.1). Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Companion Kit (ONT) and sequenced on a GridION. Reads were down-sampled to 74000 reads with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with minimap2.



Figure 2: Sequence coverage of a SARS-CoV-2 Delta clinical sample with updated primer schemes



Caption: Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (log scale). Amplicons were generated from the same Delta variant SARS-CoV-2 viral gRNA clinical sample using NEBNext® VarSkip Short SARS-CoV-2 primer pools, NEBNext VarSkip Short v2 SARS-CoV-2 primer pools, MilliporeSigma ARTICv4, MilliporeSigma ARTICv4 primer pools plus IDT ARTICv4.1 spike-in primers (ARTICv4.1). Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Companion Kit (ONT) and sequenced on GridION. Reads were down-sampled to 74000 reads with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with minimap2.


Figure 3: Express and Standard workflows for the NEBNext ARTIC SARS-CoV-2 Companion Library Prep Kit for Oxford Nanopore with VarSkip Short v2 or NEBNext ARTIC primers



 
This product is related to the following categories:
Library Preparation for Oxford Nanopore Technologies Products,
NEBNext® ARTIC products for SARS-CoV-2 sequencing,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation Products,

Properties & Usage

Materials Required but not Supplied

  • 80% Ethanol (freshly prepared)
  • DNA LoBind Tubes (Eppendorf #022431021)
  • Oxford Nanopore Technologies Native Barcoding Expansion kits 1-12 (EXP-NBD104), 13-24 (EXP-NBD114) or 1-96 (EXP-NBD196)
  • Oxford Nanopore Technologies Ligation Sequencing Kit (SQK-LSK109)
  • Oxford Nanopore Technologies SFB Expansion Kit (EXP-SFB001)
  • Qubit® dsDNA HS Assay Kit (ThermoFisher® Q32851)
  • Magnetic rack/stand (NEB #S1515, Alpaqua®, cat. #A001322 or equivalent)
  • Thermal cycler
  • Vortex Mixer
  • Microcentrifuge
  • Agilent® Bioanalyzer® or similar fragment analyzer and associated consumables (4150 or 4200 TapeStation System)
  • DNase RNase free PCR strip tubes (USA Scientific 1402-1708)
  • 1.5 ml tube magnet stand (NEB #S1506)

Features

  • Streamlined, high-efficiency protocol
  • Effective with a wide range of viral genome inputs (10-10,000 copies)
  • Improved uniformity of SARS-CoV-2 genome coverage 
  • Optional use control human primers provided
  • Includes NEBNext Sample Purification Beads (SPRIselect®)
 

References

  1. Josh Quick (2020). nCoV-2019 sequencing protocol v2 (GunIt) . protocols.io.

Protocols, Manuals & Usage

Protocols

  1. Where can I find guidelines and protocols for using the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies), including cDNA synthesis, targeted cDNA amplification, end repair and ligation, and the Express protocol option with reduced cleanup steps?

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is the difference between the NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes and the original NEBNext VarSkip Short SARS-CoV-2 Primer Mixes?
  2. What is the difference between the NEBNext VarSkip v2 Short SARS-CoV-2 Primer Mixes and the NEBNext ARTIC SARS-CoV-2 Primer Mixes?
  3. Where can I find primer sequence information for NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes and NEBNext ARTIC SARS-CoV-2 Primer Mixes?
  4. Can the NEBNext ARTIC SARS-CoV-2 Primer Mixes and NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes be added to the same targeted amplification reaction?
  5. Which primer scheme should I use: NEBNext ARTIC SARS-CoV-2 Primer Mixes or NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes?
  6. Can the NEBNext VarSkip Short v2 SARS-CoV-2 Primers cover non-variant SARS-CoV-2 samples?
  7. Which SARS-CoV-2 variants can the NEBNext VarSkip Short v2 SARS-CoV-2 Primers cover?
  8. Can the NEBNext ARTIC Human Control Primer Pairs and NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes be added to the same targeted amplification reaction?
  9. How do I analyze VarSkip Short v2 SARS-CoV-2 sequencing data?
  10. Are the sequences of the primers in the NEBNext ARTIC SARS-CoV-2 Primer Mixes the same as the ARTIC Network V3 primers?
  11. What Ct values are recommended for inputs used with the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies)?
  12. If I am using 96 samples, can I skip the cleanup step after the Targeted cDNA Amplification?
  13. How much data do I need when sequencing libraries generated using the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies)?
  14. How do I analyze my data from sequencing libraries generated using the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies)?
  15. Which kits from Oxford Nanopore Technologies do I need for the complete workflow?
  16. Is the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) compatible with the ARTIC Network nCoV-2019 sequencing protocol v3 (LoCost) or the Oxford Nanopore Technologies Eco PCR Tiling protocol?
  17. Are there tips to avoid barcode cross contamination?
  18. Is the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) compatible with all ONT instruments?
  19. Does the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) kit include sufficient beads for the entire ARTIC library prep workflow?
  20. Are there steps I can take if the pore occupancy number is low when performing sequencing?
  21. Can I use less of the native barcode adaptor during the ligation step (1.5.1)?
  22. How can the percentage of dual-barcoded reads be increased
  23. What if I don’t have enough samples to be able to generate enough library DNA for sequencing?
  24. For high Ct samples, how can the data throughput be increased?
  25. Does my total RNA sample need to be DNA-free prior to starting the ARTIC workflow?
  26. Can coverage be improved for the BA.2 variant?
  27. Is the NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) E7660 compatible with Oxford Nanopore Native Barcoding Kit V14 (SQK-NBD114)?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specification Change Notifications

Effective 6/15/2022, primer components of this product are provided in a separate box.

Effective 2/15/2022 VarSkip Short v2 SARS-CoV-2 Primer Mixes 1 and 2 replace VarSkip Short SARS-CoV-2 Primer Mixes 1 and 2.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

This product is covered by one or more patents.

This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows. For additional information or to inquire about commercial use, please contact busdev@neb.com.

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