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 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. The V3 ARTIC primers have been balanced, using methodology developed at NEB based on empirical data from sequencing, to provide uniform amplicon coverage across the viral genome.Library preparation uses the NEBNext Ultra II DNA reagents and workflow, with 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 NEBNext ARTIC SARS-CoV-2 Library Prep Kit generates libraries with inserts ~400 bp. If shorter inserts are preferred, the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (NEB #E7658) produces ~150 bp insert libraries compatible with 2 x 75 sequencing.Figure 1: Improved genome coverage with the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina)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, with or without NEBNext ARTIC Human Control Primer Pairs. Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) and sequenced on a MiSeq® instrument (2x250 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.Figure 2: Improved coverage depth per base with the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina)Amplicons were generated from 10–10,000 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 Library Prep Kit (Illumina) and sequenced on a MiSeq instrument (2x250 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.Figure 3: Including human control primers does not adversely affect genome coverageAmplicons 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 Library Prep Kit (Illumina) and sequenced on a MiSeq instrument (2x250 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.Figure 4: Express and Standard workflows for the NEBNext ARTIC SARS-CoV-2 Library Prep Kit for Illumina
This product is related to the following categories:
NEBNext? ARTIC products for SARS-CoV-2 sequencing,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation
This product can be used in the following applications:
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