Product Class: Kit

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

Information on primer overlaps with Omicron and Delta variants.

This kit does not include VarSkip primers. For improved performance with variants, VarSkip Short primers are included in the NEBNext ARTIC SARS-CoV-2 kits for smaller (~150bp) insert libraries for Illumina sequencing (NEB #E7658) and for Oxford Nanopore Technologies® sequencing (NEB #E7660), as well as in the NEBNext ARTIC SARS-CoV-2 RT-PCR Module (NEB #E7626).

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


Catalog #E7650

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. 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.  

If shorter inserts are preferred, the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) (NEB #E7658) generates libraries with ~150 bp inserts, compatible with 2 x 75 bp sequencing.

  • Information on primer variant overlaps can be found here
  • 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 with a more balanced primer pool
  • No requirement for amplicon normalization prior to library preparation
  • Library inserts in the 400 bp range
  • Compatible with 2 x 250 Illumina sequencing
  • 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

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. The V3 ARTIC primers have been balanced, using methodology developed at NEB based on empirical data from sequencing, to provide uniform amplicon coverage across the viral genome. Library preparation uses the NEBNext Ultra II DNA reagents and workflow, with 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 NEBNext ARTIC SARS-CoV-2 Library Prep Kit generates libraries with inserts ~400 bp. If shorter inserts are preferred, the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (NEB #E7658) produces ~150 bp insert libraries compatible with 2 x 75 sequencing. 

Figure 1: Improved genome coverage with the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) 



Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome. Amplicons were generated from 1,000 copies of SARS-CoV-2 viral gRNA inputs (ATCC VR-1986 and VR-1991) in 100 ng of Universal Human Reference RNA (ThermoFisher® QS0639) using IDT ARTIC nCoV-2019 V3 Panel (“Standard”) or the NEBNext balanced ARTIC SARS-CoV-2 primer pools, with or without NEBNext ARTIC Human Control Primer Pairs. Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) and sequenced on a MiSeq® instrument (2x250 bp). Coverage depth per base was determined, reads were down-sampled with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with Bowtie2.



Figure 2: Improved coverage depth per base with the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) 



Amplicons were generated from 10–10,000 copies of SARS-CoV-2 viral gRNA inputs (ATCC VR-1986 and VR-1991) in 100 ng of Universal Human Reference RNA (ThermoFisher QS0639) using IDT ARTIC nCoV-2019 V3 Panel (“Standard”) or the NEBNext balanced ARTIC SARS-CoV-2 primer pools. Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) and sequenced on a MiSeq instrument (2x250 bp). Coverage depth per base was determined, reads were down-sampled with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with Bowtie2.



Figure 3: Including human control primers does not adversely affect genome coverage



Amplicons were generated from 1,000 copies of SARS-CoV-2 viral gRNA inputs (ATCC VR-1986 and VR-1991) in 100 ng of Universal Human Reference RNA (ThermoFisher QS0639) using NEBNext balanced ARTIC SARS-CoV-2 primer pools, with or without NEBNext ARTIC Human Control Primer Pairs. Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) and sequenced on a MiSeq instrument (2x250 bp). The fraction of the genome covered at each depth was determined for a range of inputs and reads down-sampled to 10,000, 100,000, 500,000 and 1,000,000.



Figure 4: Express and Standard workflows for the NEBNext ARTIC SARS-CoV-2 Library Prep Kit for Illumina



 
 
This product is related to the following categories:
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

  • 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 with a more balanced primer pool
  • No requirement for amplicon normalization prior to library preparation
  • Library inserts in the 400 bp range
  • Compatible with 2 x 250 Illumina sequencing
  • 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 Library Prep Kit, including cDNA synthesis, cDNA amplification and library prep?

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. Are the sequences of the primers in the NEBNext ARTIC SARS-CoV-2 Primer Mixes the same as the ARTIC Network V3 primers?
  2. How many sequencing reads do I need for sequencing of libraries generated using the  NEBNext ARTIC Kits for Illumina?
  3. What Ct values are recommended for inputs used with the NEBNext ARTIC kits for Illumina?
  4. How do I analyze data from libraries generated using the NEBNext ARTIC kits for Illumina?
  5. Which NEBNext adaptors and primers are recommended for use with the ARTIC kits for Illumina?
  6. Do I need to add PhiX to the sequencing run?
  7. Do I need to normalize library concentration prior to sequencing?
  8. What read length is recommended for sequencing of libraries generated using the  NEBNext ARTIC SARS-CoV-2 Kit (Illumina)?
  9. What should the final loading concentration be for MiSeq® and NextSeq® 500/550 Illumina® sequencing platforms?
  10. Does my total RNA sample need to be DNA-free prior to starting the ARTIC workflow?

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.

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.