NEBNext® ARTIC products for SARS-CoV-2 sequencing

Especially with the ongoing emergence of SARS-CoV-2 variants that affect virus transmission and other metrics significant to public health, there is an increasing need for reliable, accurate and fast methods for sequencing SARS-CoV-2. The NEBNext ARTIC kits are based on the original work of the ARTIC Network, who quickly adapted their protocols to SARS-CoV-2 (Josh Quick 2020. nCoV-2019 sequencing protocol v2 (GunIt)).

The ARTIC method is a multiplexed amplicon-based whole-viral-genome sequencing approach (Figure 1), and the NEBNext ARTIC kit options are compatible with Illumina and Oxford Nanopore Technologies sequencing platforms. The two kits compatible with Illumina sequencing generate library inserts of ~150 bp or ~400 bp, for 2 x 75 or 2 x 250 sequencing, respectively (Figure 2). The NEBNext ARTIC SARS-CoV-2 RT-PCR Module contains only the reagents required for cDNA synthesis and targeted cDNA amplification from SARS-CoV-2 genomic RNA. 

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Figure 1: NEBNext ARTIC Workflow Overview



For more detailed workflows, use the links below:

NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) Workflow

NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) Workflow

NEBNext ARTIC SARS-CoV-2 Workflow for Oxford Nanopore Technologies Sequencing 


Figure 2: NEBNext ARTIC Kit Options



NEBNext ARTIC kits include V3 ARTIC primer pools, which have been balanced using methodology developed at NEB based on empirical data from sequencing. These balanced primers provide greater uniformity of genome coverage (Figure 3) from 10-10,000 SARS-CoV-2 genome copies. The reagents for RT-PCR and library prep are optimized for the SARS-CoV-2 ARTIC workflow.  

Figure 3: Fewer reads are required to completely cover the genome with the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) 



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 with 24500 reads or 250x data for the mapping against SARS-CoV-2 Wuhan-Hu-1.

 

Figure 4: Genome Coverage at a wide range of input amounts, 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 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. 


RT reaction conditions are the same for all input amounts, and for Illumina applications, a novel DNA polymerase formulation for the enrichment of next-generation sequencing libraries eliminates the need to normalize amplicon concentrations prior to library preparation.

  • Improved uniformity of SARS-CoV-2 genome coverage depth
  • Streamlined, high-efficiency protocols
  • Effective with a wide range of viral genome inputs (10-10,000 copies)
  • Available for Illumina and Oxford Nanopore Technologies sequencing platforms
  • Single RT conditions for all input amounts
  • No requirement for amplicon normalization prior to library preparation (Illumina-compatible kits)
  • Optional use control human primers provided
  • Includes NEBNext Sample Purification Beads (SPRIselect®)
  • Library adaptors and primers available separately

 


 
Read what leaders in ARTIC SARS-CoV-2 sequencing are saying
 

The NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) will provide researchers with the most convenient way to perform nanopore sequencing of SARS-CoV-2. When developing the ARTIC protocol we have focused on recovery of near-complete genomes from challenging clinical samples with <Ct33. We adopted a 400 bp tiling amplicon approach because it provides robust performance across a wide range of inputs including high Ct and degraded RNA samples which are often encountered. The native barcoding workflow provides full-length amplicons compatible with best practice demultiplexing and reference-based consensus building while the PCR-free library preparation minimizes amplicon contamination risk. We have worked in collaboration with NEB in optimizing the protocol to minimize reagent volumes while improving performance and this now also benefits from optimized primer pools which greatly improve genome completeness and reduce read coverage requirements. The compatibility with the 96-barcode native barcoding expansion kit allows the flexibility to run between 24 and 96 samples depending on your requirements.

- Joshua Quick, Ph.D., of the University of Birmingham and the ARTIC Network
 

We have been impressed by the great performance of the NEBNext ARTIC FS kit in our sequencing of human samples containing SARS-CoV-2 RNA. With this kit we have been successfully processing samples of varying quality and quantity and have been amazed by its sensitivity and remarkable uniformity of the viral genome coverage. The quick turnaround time of the robust workflow has been truly beneficial for us.

- Vladimir Benes, Head of Genomics Core Facility at the European Molecular Biology Laboratory
 

The NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit ensures rapid, accurate and reliable determination of the SARS-CoV-2 whole genome. Due to its ease of use, the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit enables rapid integration into the routine NGS workflow. Furthermore, the kit still works robust with difficult clinical samples (ct>30). I would recommend the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit 100%.

- Manuel Huter, Pathology Lab Dr. Obrist, Zams (Tyrol)