Next® ARTIC SARS-CoV-2 RT-PCR Module |NEB酶试剂 New England Biolabs

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产品信息

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 RT-PCR Module is based on this method and incorporates optimized reagents for the cDNA synthesis and targeted cDNA amplification steps.

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 primers:

  1. The VarSkip (for Variant Skip) Short primers have been designed at NEB to provide improved performance with SARS-CoV-2 variants, including 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.
  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.

Full kits that also include library prep reagents optimized for the ARTIC workflow are available, for smaller (~150 bp) (NEB #E7658) or larger (~400 bp) (NEB #E7650) insert libraries for Illumina® sequencing, and for Oxford Nanopore Technologies® sequencing (NEB #E7660). Note that E7650 does not include VarSkip v2 primers.

 

Figure 1: Sequence coverage of SARS-CoV-2 genomes with VarSkip Short v2 plus spike-in primers 

Next® ARTIC SARS-CoV-2 RT-PCR Module |

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

Next® ARTIC SARS-CoV-2 RT-PCR Module |

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 (2×75 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: Workflow for the NEBNext ARTIC SARS-CoV-2 RT-PCR Module with VarSkip Short v2 or NEBNext ARTIC primers 

Next® ARTIC SARS-CoV-2 RT-PCR Module |

产品类别:
NEBNext® ARTIC products for SARS-CoV-2 sequencing,
RNA Library Prep for Illumina

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E7626S     -20    
        LunaScript® RT SuperMix E7651AVIAL -20 1 x 0.048 ml 5 X
        Q5® Hot Start High-Fidelity 2X Master Mix E7652AVIAL -20 1 x 0.3 ml 2 X
        0.1X TE E7657AVIAL -20 1 x 1.3 ml Not Applicable
        Nuclease-free Water E7667AVIAL -20 1 x 1.5 ml Not Applicable
        NEBNext® ARTIC SARS-CoV-2 Primer Mix 1 E7725AVIAL -20 1 x 0.042 ml Not Applicable
        NEBNext® ARTIC SARS-CoV-2 Primer Mix 2 E7726AVIAL -20 1 x 0.042 ml Not Applicable
        NEBNext® VarSkip Short v2 SARS-CoV-2 Primer Mix 1 E7872AVIAL -20 1 x 0.042 ml Not Applicable
        NEBNext® VarSkip Short v2 SARS-CoV-2 Primer Mix 2 E7873AVIAL -20 1 x 0.042 ml Not Applicable
    • E7626L     -20    
        LunaScript® RT SuperMix E7651AAVIAL -20 1 x 0.192 ml 5 X
        Q5® Hot Start High-Fidelity 2X Master Mix E7652AAVIAL -20 1 x 1.2 ml 2 X
        0.1X TE E7657AVIAL -20 2 x 1.3 ml Not Applicable
        Nuclease-free Water E7667AVIAL -20 1 x 1.5 ml Not Applicable
        NEBNext® ARTIC SARS-CoV-2 Primer Mix 1 E7725AAVIAL -20 1 x 0.168 ml Not Applicable
        NEBNext® ARTIC SARS-CoV-2 Primer Mix 2 E7726AAVIAL -20 1 x 0.168 ml Not Applicable
        NEBNext® VarSkip Short v2 SARS-CoV-2 Primer Mix 1 E7872AAVIAL -20 1 x 0.168 ml Not Applicable
        NEBNext® VarSkip Short v2 SARS-CoV-2 Primer Mix 2 E7873AAVIAL -20 1 x 0.168 ml Not Applicable

  • 特性和用法

    需要但不提供的材料

    • 80% Ethanol (freshly prepared)
    • Nuclease-free Water
    • DNA LoBind Tubes (Eppendorf® #022431021)
    • Magnetic rack/stand (NEB #S1515, Alpaqua®, cat. #A001322 or equivalent)
    • Thermocycler
    • Vortex Mixer
    • Microcentrifuge
    • Agilent® Bioanalyzer® or similar fragment analyzer and associated consumables
    • DNase RNase free PCR strip tubes (USA Scientific®1402-1708)
    • SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)

  • 优势和特性

    Features

    • Improved uniformity of SARS-CoV-2 genome coverage depth with a more balanced primer pool
    • Effective with a wide range of viral genome inputs (10-10,000 copies)
    • Streamlined, high-efficiency protocol

  • 相关产品

    相关产品

    • Monarch® 总 RNA 小量提取试剂盒
    • NEBNext® 多样本接头引物试剂盒 1（96 种 Unique 双端 Index 引物）
    • NEBNext® 多样本接头引物试剂盒 2（96 种 Unique 双端 Index 引物）
    • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3)

  • 参考文献

    1. Josh Quick (2020). nCoV-2019 sequencing protocol v2 (GunIt). protocols.io.

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find guidelines and protocols for using the NEBNext ARTIC SARS-CoV-2 RT-PCR Module, including cDNA synthesis and cDNA amplification?

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE7626 with VarSkip Short SARS-CoV-2 Primers v2
    • manualE7626 with VarSkip Short v2 SARS-CoV-2 Primers v3

工具 & 资源

  • Web 工具

    • NEBNext Selector

FAQs & 问题解决指南

  • FAQs

    1. What is the difference between the NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes and the original NEBNext VarSkip Short SARS-CoV-2 Primer Mixes?
    2. What is the difference between the NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes and the NEBNext ARTIC SARS-CoV-2 Primer Mixes?
    3. Where can I find primer sequence information for NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes and NEBNext ARTIC SARS-CoV-2 Primer Mixes?
    4. Can the NEBNext ARTIC SARS-CoV-2 Primer Mixes and NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes be added to the same targeted amplification reaction?
    5. Which primer scheme should I use: NEBNext ARTIC SARS-CoV-2 Primer Mixes or NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes?
    6. Can the NEBNext VarSkip Short v2 SARS-CoV-2 Primers cover non-variant SARS-CoV-2 samples?
    7. Which SARS-CoV-2 variants can the NEBNext VarSkip Short v2 SARS-CoV-2 Primers cover?
    8. How do I analyze VarSkip Short v2 SARS-CoV-2 sequencing data?
    9. Does my total RNA sample need to be DNA-free prior to starting the ARTIC workflow?
    10. Can coverage be improved for the BA.2 variant?