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.This kit includes two options for primers, including new VarSkip Short v2 primers:
The VarSkip (for Variant Skip) Short primers have been designed at NEB to provide improved performance with SARS-CoV-2 variants, including 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 foundhere.
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.
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).Note that E7650 does not include VarSkip v2 primers.
Figure 1: Sequence coverage of SARS-CoV-2 genomes with VarSkip Short v2 plus spike-in primersConsensus 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 schemesIntegrative 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: Workflow for the NEBNext ARTIC SARS-CoV-2 RT-PCR Module with VarSkip Short v2 or NEBNext ARTIC primers
This product is related to the following categories:
NEBNext® ARTIC products for SARS-CoV-2 sequencing,
RNA Library Prep for Illumina
This product can be used in the following applications:
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