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OXFORD NANOPORE MIDNIGHT PROTOCOL PLUS

Reads were down-sampled to 74000 reads with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with minimap2. Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Companion Kit (ONT) and sequenced on a GridION.

OXFORD NANOPORE MIDNIGHT PROTOCOL PLUS

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 VarSkip Short v2 SARS-CoV-2 primer pools, MilliporeSigma ARTICv4, MilliporeSigma ARTICv4 primer pools plus IDT ARTICv4.1 spike-in primers (ARTICv4.1). 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.įigure 4: Coverage of a SARS-CoV-2 Omicron clinical sample with updated primer schemes and Oxford Nanopore Technologies sequencingĬaption: Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (log scale). Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina ®) and sequenced on a MiSeq ® instrument (2x75 bp). The same Omicron variant SARS-CoV-2 viral gRNA clinical sample served as a template for all primer schemes except ARTICv3. 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. Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (log scale). The reagents for RT-PCR and library prep are optimized for the SARS-CoV-2 ARTIC workflow.įigure 3: Coverage of a SARS-CoV-2 Omicron clinical sample with updated primer schemes and Illumina sequencing These balanced primers provide greater uniformity of genome coverage (Figure 3) from 10-10,000 SARS-CoV-2 genome copies. NEBNext ARTIC kits include V3 ARTIC primer pools, which have been balanced using methodology developed at NEB based on empirical data from sequencing. NEBNext ARTIC SARS-CoV-2 Workflow for Oxford Nanopore Technologies Sequencing NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) Workflow

NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) Workflow Express protocols are also available, with fewer clean ups.įor more detailed workflows, use the links below: In combination with optimized reagents for RT-PCR, the kits deliver improved uniformity of amplicon yields from gRNA across a wide copy number range.įigure 1: NEBNext ARTIC Standard Workflow Overview.

The V3 ARTIC primers have been balanced, using methodology developed at NEB based on empirical data from sequencing.

Information on primer overlaps with Omicron and Delta variants. The primer pools are balanced for coverage uniformity.

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 variant.

Two options for primers are now available, including new VarSkip Short v2 primers, 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). 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.Įxpress workflow options are provided, with reduced cleanup steps.

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. nCoV-2019 sequencing protocol v2 (GunIt)). 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. 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.