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.
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®)
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
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Additional Citations
Peter AS, et al. (2021) A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model. bioRxiv; DOI: 10.1101/2021.04.16.440101
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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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