Product Class: Kit

NEBNext® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®)

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 spike-in primers. 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 #E7658

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, for improved Omicron coverage:

  1. See below, including Figure 1 in the Product Information section, for information on VarSkip Short v2 performance with spike-in primers. 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. 

An Express protocol option with fewer cleanups is available.

The NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) incorporates enzymatic cDNA fragmentation and produces Illumina-compatible libraries with library inserts in the 150 bp range, compatible with 2 x 75 sequencing on Illumina instruments.

  • Includes two primer options: new VarSkip Short v2, for improved Omicron coverage, and 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)
  • No requirement for amplicon normalization prior to library preparation
  • Library inserts in the 150 bp range
  • Compatible with 2 x 75 Illumina sequencing
  • Optional use control human primers provided
  • Includes NEBNext Sample Purification Beads (SPRIselect®)
  • Adaptors and primers available separately; For a 384 unique dual index format, please contact us
  • For a bulk format of the kit (2,304 reactions) please contact us

If larger inserts are preferred, the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) (NEB #E7650) generates libraries with ~400 bp inserts, compatible with 2 x 250 bp sequencing.

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 FS 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, for improved Omicron coverage

  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. 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.

Library preparation uses the NEBNext FS reagents and workflow, incorporating enzymatic cDNA fragmentation, and reagent volumes tailored to the ARTIC application. Note that adaptors and primers for library prep are purchased separately. The novel NEBNext Library PCR Master Mix allows use of the same number of PCR cycles to amplify all libraries, regardless of initial input amounts. 

The Express protocol option removes the cleanup of cDNA amplicons and the cleanup of adaptor-ligated DNA. The Express protocol is recommended for inputs of ≥ 100 copies of the (SARS-CoV-2) viral genome.

The NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit generates libraries with inserts ~150 bp. If longer inserts are preferred, the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (NEB E7650) produces ~400 bp insert libraries, compatible with 2 x 250 Illumina sequencing. Note that this kit does not contain VarSkip v2 primers. For Oxford Nanopore Technologies® sequencing the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) (NEB #E7660) can be used. 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.

 

Figure 1: Sequence coverage of SARS-CoV-2 genomes with VarSkip Short v2 plus spike-in primers 



Consensus genomes known to be generated using VarSkip Short 2 (+ supplementary primers) (green) are compared with other genomes (yellow) submitted to Genbank between 2022-08-01 and 2022-09-24. Genomes were aligned to the NC_045512.2 reference (minimap2 -x map-ont -r 20000 --score-N=0, v.2.24). Ns were tabulated using Qualimap (v2.2.2).



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



Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (log scale). Amplicons were generated using NEBNext® VarSkip Short SARS-CoV-2 v2 primer pools, NEBNext VarSkip Short SARS-CoV-2 primer pools, NEBNext ARTIC SARS-CoV-2 primer pools (ARTICv3), MilliporeSigma ARTICv4 primer pools plus IDT ARTICv4.1 spike-in primers (ARTICv4.1), or MilliporeSigma ARTICv4 primer pools. The same Omicron variant SARS-CoV-2 viral gRNA clinical sample served as a template for all primer schemes except ARTICv3. Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) and sequenced on a MiSeq® instrument (2x75 bp). Coverage depth per base was determined, reads were down-sampled to 0.5M PE reads with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with Bowtie2.




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



This product is related to the following categories:
NEBNext® ARTIC products for SARS-CoV-2 sequencing

Properties & Usage

Materials Required but not Supplied

  • NEBNext Singleplex or Multiplex Oligos for Illumina –www.neb.com/oligos
  • 80% Ethanol (freshly prepared)
  • Nuclease-free Water
  • DNA LoBind Tubes (Eppendorf®#022431021)
  • Magnetic rack/stand (NEB #S1515, Alpaqua®, cat. #A001322 or equivalent)
  • Thermocycler
  • Vortex Mixer
  • Microcentrifuge
  • Agilent®Bioanalyzer®or similar fragment analyzer and associated consumables
  • DNase RNase free PCR strip tubes (USA Scientific®1402-1708)

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 depth
  • No requirement for amplicon normalization prior to library preparation
  • Library inserts in the 150 bp range
  • Compatible with 2 x 75 Illumina sequencing
  • Optional use control human primers provided
  • Includes NEBNext Sample Purification Beads (SPRIselect®)
  • Adaptors and primers available separately; For a 384 unique dual index format, please contact us

References

  1. Josh Quick 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 FS Library Prep Kit, including cDNA synthesis, cDNA amplification and library prep, 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 read length is recommended for sequencing of libraries generated using the  NEBNext ARTIC SARS-CoV-2 FS Kit (Illumina)?
  12. How many sequencing reads do I need for sequencing of libraries generated using the  NEBNext ARTIC Kits for Illumina?
  13. What Ct values are recommended for inputs used with the NEBNext ARTIC kits for Illumina?
  14. How do I analyze data from libraries generated using the NEBNext ARTIC kits for Illumina?
  15. Which NEBNext adaptors and primers are recommended for use with the ARTIC kits for Illumina?
  16. Do I need to add PhiX to the sequencing run?
  17. Do I need to normalize library concentration prior to sequencing?
  18. What should the final loading concentration be for MiSeq® and NextSeq® 500/550 Illumina® sequencing platforms?
  19. Does my total RNA sample need to be DNA-free prior to starting the ARTIC workflow?
  20. Can coverage be improved for the BA.2 variant?

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 [email protected].

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 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.

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