Next® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) |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 FS 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.

This kit includes two options for primers, including new VarSkip Short v2, for improved Omicron coverage

  1. The new VarSkip (for Variant Skip) Short v2 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. 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.

Library preparation uses the NEBNext FS reagents and workflow, incorporating enzymatic cDNA fragmentation, and 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 Express protocol option removes the cleanup of cDNA amplicons and the cleanup of adaptor-ligated DNA. The Express protocol is recommended for inputs of ≥ 100 copies of the (SARS-CoV-2) viral genome.

The NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit generates libraries with inserts ~150 bp. If longer inserts are preferred, the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (NEB E7650) produces ~400 bp insert libraries, compatible with 2 x 250 Illumina sequencing. Note that this kit does not contain VarSkip v2 primers. For Oxford Nanopore Technologies® sequencing the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) (NEB #E7660) can be used. If only the reagents for cDNA synthesis and targeted cDNA amplification are required, the NEBNext ARTIC SARS-CoV-2 RT-PCR Module (NEB #E7626) is available.

 

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

Next® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) |

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 FS Library Prep Kit (Illumina®) |

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: Express and Standard workflows for the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit for Illumina with VarSkip Short v2 or NEBNext ARTIC primers 

Next® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) |

产品类别:
NEBNext® ARTIC products for SARS-CoV-2 sequencing

  • 试剂盒组成

    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
    • E7658S     Multi-temperature    
        NEBNext® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) E7658-1 -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
        NEBNext® Ultra II™ FS Enzyme Mix E7668AVIAL -20 1 x 0.024 ml Not Applicable
        NEBNext® Ultra II™ FS Reaction Buffer E7669AVIAL -20 1 x 0.084 ml Not Applicable
        NEBNext® Ultra II™Ligation Master Mix E7655AVIAL -20 1 x 0.36 ml Not Applicable
        NEBNext® Library PCR Master Mix E7656AVIAL -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® Sample Purification Beads E7659S 25    
        NEBNext® Sample Purification Beads E7659SVIAL 25 1 x 2.1 ml Not Applicable
        NEBNext® ARTIC SARS-CoV-2 Primer Mixes E7871S -20    
        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® ARTIC Human Control Primer Pairs 1 E7727AVIAL -20 1 x 0.007 ml Not Applicable
        NEBNext® ARTIC Human Control Primer Pairs 2 E7728AVIAL -20 1 x 0.007 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
    • E7658L     Multi-temperature    
        NEBNext® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) E7658-2 -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
        NEBNext® Ultra II™ FS Enzyme Mix E7668AAVIAL -20 1 x 0.096 ml Not Applicable
        NEBNext® Ultra II™ FS Reaction Buffer E7669AAVIAL -20 1 x 0.336 ml Not Applicable
        NEBNext® Ultra II™Ligation Master Mix E7655AAVIAL -20 2 x 0.72 ml Not Applicable
        NEBNext® Library PCR Master Mix E7656AAVIAL -20 1 x 1.2 ml 2 X
        0.1X TE E7657AAVIAL -20 1 x 5.2 ml Not Applicable
        Nuclease-free Water E7667AVIAL -20 1 x 1.5 ml Not Applicable
        NEBNext® Sample Purification Beads E7659L 25    
        NEBNext® Sample Purification Beads E7659SVIAL 25 4 x 2.1 ml Not Applicable
        NEBNext® ARTIC SARS-CoV-2 Primer Mixes E7871L -20    
        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® ARTIC Human Control Primer Pairs 1 E7727AVIAL -20 1 x 0.007 ml Not Applicable
        NEBNext® ARTIC Human Control Primer Pairs 2 E7728AVIAL -20 1 x 0.007 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

    • NEBNext Singleplex or Multiplex Oligos for Illumina –www.neb.com/oligos
    • 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)

  • 优势和特性

    Features

    • Streamlined, high-efficiency protocol
    • Effective with a wide range of viral genome inputs (10-10,000 copies)
    • Improved uniformity of SARS-CoV-2 genome coverage depth
    • No requirement for amplicon normalization prior to library preparation
    • Library inserts in the 150 bp range
    • Compatible with 2 x 75 Illumina sequencing
    • Optional use control human primers provided
    • Includes NEBNext Sample Purification Beads (SPRIselect®)
    • Adaptors and primers available separately; For a 384 unique dual index format, please contact us

  • 相关产品

    相关产品

    • E7650 NEBNext ARTIC SARS-CoV-2 Library Prep Kit Illumina
    • E7660 NEBNext ARTIC SARS-CoV-2 Companion Kit Oxford Nanopore Technologies

  • 参考文献

    1. Josh Quick 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 FS Library Prep Kit, including cDNA synthesis, cDNA amplification and library prep, and the Express protocol option with reduced cleanup steps?

  • 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。

    • manualE7658 with VarSkip Short SARS-Co-V2 Primers v4point1
    • manualE7658 with VarSkip Short v2 SARS-CoV-2 Primers v5

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. Can the NEBNext ARTIC Human Control Primer Pairs and NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes be added to the same targeted amplification reaction?
    9. How do I analyze VarSkip Short v2 SARS-CoV-2 sequencing data?
    10. Are the sequences of the primers in the NEBNext ARTIC SARS-CoV-2 Primer Mixes the same as the ARTIC Network V3 primers?
    11. What read length is recommended for sequencing of libraries generated using the  NEBNext ARTIC SARS-CoV-2 FS Kit (Illumina)?
    12. How many sequencing reads do I need for sequencing of libraries generated using the  NEBNext ARTIC Kits for Illumina?
    13. What Ct values are recommended for inputs used with the NEBNext ARTIC kits for Illumina?
    14. How do I analyze data from libraries generated using the NEBNext ARTIC kits for Illumina?
    15. Which NEBNext adaptors and primers are recommended for use with the ARTIC kits for Illumina?
    16. Do I need to add PhiX to the sequencing run?
    17. Do I need to normalize library concentration prior to sequencing?
    18. What should the final loading concentration be for MiSeq® and NextSeq® 500/550 Illumina® sequencing platforms?
    19. Does my total RNA sample need to be DNA-free prior to starting the ARTIC workflow?
    20. Can coverage be improved for the BA.2 variant?