NEBNext ARTIC SARS-CoV-2 kits are also available for larger (~400bp) insert libraries for Illumina sequencing (NEB #E7650), and for Oxford Nanopore Technologies® sequencing (NEB #E7660).
Catalog #E7658
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 balanced V3 primers and optimized reagents for RT-PCR deliver improved uniformity of amplicon yields from gRNA across a wide copy number range.
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.
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.
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
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®)
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. The multiplex primers are balanced to provide uniform amplicon coverage across the viral genome. 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 NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit generates libraries with inserts ~250 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 sequencing.
Figure 1: Improved genome coverage with the NEBNext ARTIC SARS-CoV-2 FS 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 FS Library Prep Kit (Illumina) and sequenced on a MiSeq® instrument (2x75 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 NEBNext ARTIC SARS-CoV-2 FS 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 FS 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 FS Library Prep Kit (Illumina) and sequenced on a MiSeq instrument (2x75 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: Improved genome coverage with the NEBNext ARTIC SARS-CoV-2 FS 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 QIAseq SARS-CoV-2 Primer Panel or the NEBNext balanced ARTIC SARS-CoV-2 primer pools. Libraries were constructed using the QIAseqFX kit or 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 with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with Bowtie2.
Figure 5: Improved genome coverage with the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina)
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 QIAseq SARS-CoV-2 Primer Panel or the NEBNext balanced ARTIC SARS-CoV-2 primer pools. Libraries were constructed using the QIAseqFX kit or the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) and sequenced on a MiSeq instrument (2x75 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 6: NEBNext ARTIC SARS-CoV-2 FS DNA (Illumina) Workflow
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For more information about commercial rights, please contact NEB's Global Business Development team at [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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