标签归档：artic

Next® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

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 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 primers:

  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 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.

The Express protocol removes the cleanup of cDNA Amplicons.

The NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) is compatible with sequencing on the Oxford Nanopore Technologies platform and is used alongside kits from Oxford Nanopore Technologies to generate libraries with inserts in the  400bp range, that can be sequenced on any Oxford Nanopore Technologies instrument, in 2-24 hour run times.

If Illumina-compatible libraries are preferred, the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) (NEB #E7658) and the NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina) (NEB #E7650) generate libraries with ~150 bp and ~400 bp inserts, respectively. 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.  Note that E7650 does not include VarSkip v2 primers.

Figure 1: Sequence coverage of a SARS-CoV-2 Omicron clinical sample with updated primer schemes

Next® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) |

Caption: 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 VarSkip Short v2 SARS-CoV-2 primer pools, MilliporeSigma ARTICv4, MilliporeSigma ARTICv4 primer pools plus IDT ARTICv4.1 spike-in primers (ARTICv4.1). Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Companion Kit (ONT) and sequenced on a GridION. Reads were down-sampled to 74000 reads with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with minimap2.
Figure 2: Sequence coverage of a SARS-CoV-2 Delta clinical sample with updated primer schemes

Next® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) |

Caption: Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (log scale). Amplicons were generated from the same Delta 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). Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Companion Kit (ONT) and sequenced on GridION. Reads were down-sampled to 74000 reads with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with minimap2.
Figure 3: Express and Standard workflows for the NEBNext ARTIC SARS-CoV-2 Companion Library Prep Kit for Oxford Nanopore with VarSkip Short v2 or NEBNext ARTIC primers

Next® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) |

 
产品类别:
NEBNext® ARTIC products for SARS-CoV-2 sequencing,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation Products

  • 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
    • E7660S     Multi-temperature    
        NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) E7660-1 -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™ End Prep Enzyme Mix E7661AVIAL -20 1 x 0.018 ml Not Applicable
        NEBNext® Ultra II™ End Prep Buffer E7662AVIAL -20 1 x 0.042 ml Not Applicable
        Blunt/TA Ligase Master Mix E7663AVIAL -20 1 x 0.24 ml Not Applicable
        NEBNext® Quick T4 DNA Ligase E7664AVIAL -20 1 x 0.02 ml Not Applicable
        NEBNext® Quick Ligation Reaction Buffer E7665AVIAL -20 1 x 0.04 ml 5 X
        Nuclease-free Water E7667AVIAL -20 1 x 1.5 ml Not Applicable
        NEBNext® Sample Purification Beads E7666S 25    
        NEBNext® Sample Purification Beads E7666SVIAL 25 1 x 0.872 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
    • E7660L     Multi-temperature    
        NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) E7660-2 -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™ End Prep Enzyme Mix E7661AAVIAL -20 1 x 0.072 ml Not Applicable
        NEBNext® Ultra II™ End Prep Buffer E7662AAVIAL -20 1 x 0.168 ml Not Applicable
        Blunt/TA Ligase Master Mix E7663AAVIAL -20 1 x 0.96 ml Not Applicable
        NEBNext® Quick T4 DNA Ligase E7664AVIAL -20 1 x 0.02 ml Not Applicable
        NEBNext® Quick Ligation Reaction Buffer E7665AVIAL -20 1 x 0.04 ml 5 X
        Nuclease-free Water E7667AAVIAL -20 1 x 4.7 ml Not Applicable
        NEBNext® Sample Purification Beads E7666L 25    
        NEBNext® Sample Purification Beads E7666LVIAL 25 1 x 2.9 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

    • 80% Ethanol (freshly prepared)
    • DNA LoBind Tubes (Eppendorf #022431021)
    • Oxford Nanopore Technologies Native Barcoding Expansion kits 1-12 (EXP-NBD104), 13-24 (EXP-NBD114) or 1-96 (EXP-NBD196)
    • Oxford Nanopore Technologies Ligation Sequencing Kit (SQK-LSK109)
    • Oxford Nanopore Technologies SFB Expansion Kit (EXP-SFB001)
    • Qubit® dsDNA HS Assay Kit (ThermoFisher® Q32851)
    • Magnetic rack/stand (NEB #S1515, Alpaqua®, cat. #A001322 or equivalent)
    • Thermal cycler
    • Vortex Mixer
    • Microcentrifuge
    • Agilent® Bioanalyzer® or similar fragment analyzer and associated consumables (4150 or 4200 TapeStation System)
    • DNase RNase free PCR strip tubes (USA Scientific 1402-1708)
    • 1.5 ml tube magnet stand (NEB #S1506)

  • 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 
    • Optional use control human primers provided
    • Includes NEBNext Sample Purification Beads (SPRIselect®)
     

  • 相关产品

    相关产品

    • E7658 NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit Illumina
    • E7650 NEBNext ARTIC SARS-CoV-2 Library Prep Kit Illumina

  • 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 Companion Kit (Oxford Nanopore Technologies), including cDNA synthesis, targeted cDNA amplification, end repair and ligation, 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。

    • manualE7660 with VarSkip Short SARS-CoV-2 Primers v5
    • manualE7660 with VarSkip Short v2 SARS-CoV-2 Primers v6
    • manualE7660 with VarSkip Short v2 SARS-CoV-2 Primers v7

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 Ct values are recommended for inputs used with the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies)?
    12. If I am using 96 samples, can I skip the cleanup step after the Targeted cDNA Amplification?
    13. How much data do I need when sequencing libraries generated using the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies)?
    14. How do I analyze my data from sequencing libraries generated using the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies)?
    15. Which kits from Oxford Nanopore Technologies do I need for the complete workflow?
    16. Is the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) compatible with the ARTIC Network nCoV-2019 sequencing protocol v3 (LoCost) or the Oxford Nanopore Technologies Eco PCR Tiling protocol?
    17. Are there tips to avoid barcode cross contamination?
    18. Is the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) compatible with all ONT instruments?
    19. Does the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) kit include sufficient beads for the entire ARTIC library prep workflow?
    20. Are there steps I can take if the pore occupancy number is low when performing sequencing?
    21. Can I use less of the native barcode adaptor during the ligation step (1.5.1)?
    22. How can the percentage of dual-barcoded reads be increased?
    23. What if I don’t have enough samples to be able to generate enough library DNA for sequencing?
    24. For high Ct samples, how can the data throughput be increased?
    25. Does my total RNA sample need to be DNA-free prior to starting the ARTIC workflow?
    26. Can coverage be improved for the BA.2 variant?
    27. Is the NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) E7660 compatible with Oxford Nanopore Native Barcoding Kit V14 (SQK-NBD114)?

Next® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

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 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) 
Next® 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 (2×250 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) 

Next® 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 (2×250 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 coverage
Next® ARTIC SARS-CoV-2 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 Library Prep Kit (Illumina) and sequenced on a MiSeq instrument (2×250 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

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

 
 
产品类别:
NEBNext® ARTIC products for SARS-CoV-2 sequencing,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation Products

  • 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
    • E7650S     Multi-temperature    
        NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) E7650-1 -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™ End Prep Enzyme Mix E7653AVIAL -20 1 x 0.036 ml Not Applicable
        NEBNext® Ultra II™ End Prep Buffer E7654AVIAL -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® 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® Sample Purification Beads E7659S 25    
        NEBNext® Sample Purification Beads E7659SVIAL 25 1 x 2.1 ml Not Applicable
    • E7650L     Multi-temperature    
        NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) E7650-2 -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™ End Prep Enzyme Mix E7653AAVIAL -20 1 x 0.144 ml Not Applicable
        NEBNext® Ultra II™ End Prep Buffer E7654AAVIAL -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® 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® Sample Purification Beads E7659L 25    
        NEBNext® Sample Purification Beads E7659SVIAL 25 4 x 2.1 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 with a more balanced primer pool
    • No requirement for amplicon normalization prior to library preparation
    • Library inserts in the 400 bp range
    • Compatible with 2 x 250 Illumina sequencing
    • 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

    • E7658 NEBNext ARTIC SARS-CoV-2 FS 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 Library Prep Kit, including cDNA synthesis, cDNA amplification and library prep?

  • 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。

    • manualE7650

工具 & 资源

  • Web 工具

    • NEBNext Selector

FAQs & 问题解决指南

  • FAQs

    1. Are the sequences of the primers in the NEBNext ARTIC SARS-CoV-2 Primer Mixes the same as the ARTIC Network V3 primers?
    2. How many sequencing reads do I need for sequencing of libraries generated using the  NEBNext ARTIC Kits for Illumina?
    3. What Ct values are recommended for inputs used with the NEBNext ARTIC kits for Illumina?
    4. How do I analyze data from libraries generated using the NEBNext ARTIC kits for Illumina?
    5. Which NEBNext adaptors and primers are recommended for use with the ARTIC kits for Illumina?
    6. Do I need to add PhiX to the sequencing run?
    7. Do I need to normalize library concentration prior to sequencing?
    8. What read length is recommended for sequencing of libraries generated using the  NEBNext ARTIC SARS-CoV-2 Kit (Illumina)?
    9. What should the final loading concentration be for MiSeq® and NextSeq® 500/550 Illumina® sequencing platforms?
    10. Does my total RNA sample need to be DNA-free prior to starting the ARTIC workflow?

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

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

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?

Next® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

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?