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. 

Express workflow options are provided, with reduced cleanup steps.

Two options for primers are now available, in the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) (NEB #E7658), NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) (NEB #E7660) and NEBNext ARTIC SARS-CoV-2 RT-PCR Module (NEB #E7626).

  1. The VarSkip (for Variant Skip) Short 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 and Delta variants. The Omicron variant can be called confidently using NEBNext VarSkip Short (VSS) primers. Note that there are two dropouts (amplicons 56 and 67), and two amplicons (20 and 64) have lower coverage. Making 2 libraries, with both ARTIC V3 and VSS, will provide near complete genome coverage. The primer pools are balanced for coverage uniformity. 

    Information on primer overlaps with Omicron and Delta variants.

  2. 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. 

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Figure 1: NEBNext ARTIC Standard Workflow Overview. Express protocols are also available, with fewer clean ups.



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: Sequence coverage of a SARS-CoV-2 Omicron clinical sample with different primer schemes



Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (log scale). Amplicons were generated from the same Omicron variant SARS-CoV-2 viral gRNA clinical sample using NEBNext VarSkip Short SARS-CoV-2 primer pools, NEBNext ARTIC SARS-CoV-2 primer pools (ARTICv3), Sigma ARTICv4, or IDT Midnight-1200 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 to 0.5M PE reads with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with Bowtie2.


Figure 4: Improved sequence coverage of SARS-CoV-2 Delta clinical samples with VarSkip Short primers 



Consensus sequences classified as B.1.617.2 (Delta) in Massachusetts (where VarSkip Short was field tested) were collected from NCBI Virus [1] and partitioned into 2,613 ARTIC V3 and 1,319 VarSkip Short sequences using the SRA “design” field (NEB_VarSkip_v1 = VarSkip Short, others assumed to be ARTIC V3). Reads were aligned to the NC_045512.2 reference using minimap2 (minimap2 -x asm5) and visualized using IGV. Consensus sequences generated from VarSkip amplicons suffered fewer dropouts.
 
[1] NCBI virus link to data

Figure 5: Improved SARS-CoV-2 variant genome coverage with NEBNext VarSkip Short Primers 



Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (log scale). Amplicons were generated from 1,000 copies of standard or variant SARS-CoV-2 viral gRNA controls (ATCC VR-1986; Twist Synthetic SARS-CoV-2 RNA Control 16, 17, 18, and 23) in 100 ng of Universal Human Reference RNA (ThermoFisher® QS0639) using NEBNext ARTIC SARS-CoV-2 primer pools or NEBNext VarSkip Short 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.

Note that Twist SARS-CoV-2 controls consists of six non-overlapping 5 kb fragments of ssRNA. Primer pairs that lie on different fragments will not generate amplicons, and in IGV plots this results in ”gaps” in coverage approximately every 5kb.


Figure 6: 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 7: 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.

  • Two primer options included: VarSkip Short or ARTIC V3
  • Express protocols available, with reduced cleanup steps
  • Improved uniformity of SARS-CoV-2 genome coverage depth with balanced primer pools
  • 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
  • Bulk formats (2,304 reactions) also available. Please contact us

 


 
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)