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.
This kit includes two options for primers, including new VarSkip Short v2, for improved Omicron coverage
The new VarSkip (for Variant Skip) Short v2 primers have been designed at NEB to reduce the impact of SARS-CoV-2 variants on amplification and provide improved performance, including with 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 found here.
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.
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 Express protocol option removes the cleanup of cDNA amplicons and the cleanup of adaptor-ligated DNA. The Express protocol is recommended for inputs of ≥ 100 copies of the (SARS-CoV-2) viral genome.
The NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit generates libraries with inserts ~150 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 Illumina sequencing. Note that this kit does not contain VarSkip v2 primers. For Oxford Nanopore Technologies® sequencing the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) (NEB #E7660) can be used. If only the reagents for cDNA synthesis and targeted cDNA amplification are required, the NEBNext ARTIC SARS-CoV-2 RT-PCR Module (NEB #E7626) is available.
Figure 1: Sequence coverage of SARS-CoV-2 genomes with VarSkip Short v2 plus spike-in primers
Consensus 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 schemes
Integrative 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: Express and Standard workflows for the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit for Illumina with VarSkip Short v2 or NEBNext ARTIC primers
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Additional Citations
Mallm J-P, et al. (2021) Local emergence and decline of a SARS-CoV-2 variant with mutations L452R and N501Y in the spike protein. medRxiv; DOI: 10.1101/2021.04.27.21254849
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