NEBNext® ARTIC SARS-CoV-2 RT-PCR Module

NEBNext ARTIC SARS-CoV-2 kits that also include library prep reagents are available for smaller (~150 bp) (NEB #E7658) or larger (~400 bp) (NEB #E7650) insert libraries for Illumina® sequencing, and for Oxford Nanopore Technologies® sequencing (NEB #E7660).

 

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

NEBNext ARTIC SARS-CoV-2 RT-PCR Module contains the reagents required for cDNA synthesis and targeted cDNA amplification from SARS-CoV-2 genomic RNA. The balanced NEBNext ARTIC primers and optimized reagents deliver improved uniformity of amplicon yields from gRNA across a wide copy number range. 

Full kits that also include library prep reagents optimized for the ARTIC workflow are available, for smaller (~150 bp) (NEB #E7658) or larger (~400 bp) (NEB #E7650) insert libraries for Illumina® sequencing, and for Oxford Nanopore Technologies® sequencing (NEB #E7660).

  • Improved uniformity of SARS-CoV-2 genome coverage depth with a more balanced primer pool
  • Effective with a wide range of viral genome inputs (10-10,000 copies)
  • Streamlined, high-efficiency protocol

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

Catalog # Concentration Size
E7626S Not Applicable 24 reactions
E7626L Not Applicable 96 reactions
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  • Product Information
    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 RT-PCR Module is based on this method and incorporates optimized reagents for the cDNA synthesis and targeted cDNA amplification steps.

    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. 

    Full kits that also include library prep reagents optimized for the ARTIC workflow are available, for smaller (~150 bp) (NEB #E7658) or larger (~400 bp) (NEB #E7650) insert libraries for Illumina® sequencing, and for Oxford Nanopore Technologies® sequencing (NEB #E7660).

    Figure 1: Robust SARS-CoV-2 RT-PCR Amplification



    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 NEBNext balanced ARTIC SARS-CoV-2 primer pools. Mean amplicon yield was measured using the Qubit™ dsDNA BR Assay Kit (ThermoFisher Q32850).



    Figure 2: Improved genome coverage with the NEBNext ARTIC SARS-CoV-2 Primer Pools and Illumina sequencing



    Integrative 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. 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 3: Improved genome coverage with fewer reads with the NEBNext ARTIC SARS-CoV-2 Primer Pools and Oxford Nanopore Technologies sequencing



    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. Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) and the Oxford Nanopore Technologies Native Barcoding Expansion kits 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114), Ligation Sequencing Kit (SQK-LSK109) and SFB Expansion Kit (EXP-SFB001). Sequencing was on a GridION instrument using R9.4.1 flow cells. Minimap2 was used.




    Figure 4: NEBNext ARTIC SARS-CoV-2 RT-PCR Module Workflow 




    Reagents Supplied

    The following reagents are supplied with this product:

    NEB # Component Name Component # Stored at (°C) Amount Concentration
    • E7626S     -20    
    • E7626L     -20    
    Product Categories:
    RNA Library Prep Products,
    NGS Sample Prep & Target Enrichment Products
    Applications:
    NEBNext® ARTIC products for SARS-CoV-2 sequencing,
    RNA Library Preparation,
    RNA For Illumina,
    RNA-seq,
    RNA Analysis,
    NGS Sample Prep & Target Enrichment,
    Illumina Library Preparation
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