Next Direct® Custom Ready Panels |NEB酶试剂 New England Biolabs

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NEBNext Direct® Custom Ready Panels

Flexible Precision.

Employing the unique NEBNext Direct hybridization-based enrichment method, NEBNext Direct Custom Ready Panels allow rapid customization of targeted gene panels for Illumina® sequencing. Select from a list of human genes where baits have been carefully designed and optimized to provide complete coverage of the full coding (exon) regions. High quality panels can be designed by you and rapidly delivered, from any combination of genes. NEBNext Direct Custom Ready Panels provide the content you want with the performance you need.

  • Choose from a single gene to hundreds of genes. View Ordering Guide.  
  • Experience unmatched specificity and coverage uniformity
  • Eliminate synthesis and optimization steps, for a faster turnaround
  • Improve sensitivity with our Unique Molecular Identifier (UMI)
  • Generate results in one day with our automation-friendly workflow

Next Direct® Custom Ready Panels |

产品信息

Select your genes from our extensive library! 

Target enrichment, coupled with next generation sequencing (NGS), enables high-throughput, deep sequencing of genomic regions of interest. NEBNext Direct is a novel, hybridization-based capture method offering significant advantages over traditional in-solution hybridization and multiplex PCR protocols.

In the NEBNext Direct target enrichment approach (Figure 1), enzymatically nicked or Covaris® sheared DNA is hybridized to biotinylated oligonucleotide baits that capture both strands of the target DNA and define the 3´ ends of the regions of interest. After hybridization, the bait-target hybrids are bound to streptavidin beads and any 3´ off-target sequence is removed enzymatically. This combination of hybridization with enzymatic removal of 3´ off-target sequence enables greater sequencing specificity relative to conventional hybridization-based enrichment methods. The trimmed targets are then converted into Illumina-compatible libraries that include a 12 bp unique molecular identifier (UMI) in the Illumina i5 index location and an 8 bp sample barcode in the Illumina i7 index location. The NEBNext Direct enrichment method can be performed within one to two days and is compatible with most automated liquid handling instruments.

The NEBNext Direct Custom Ready Panels are designed to enrich for the complete exonic content of genes selected from the Custom Ready portfolio for next generation sequencing on the Illumina platform. This kit contains the oligonucleotides, beads, enzymes and buffers required to convert the desired fragments into a sequence-ready library. Custom Ready Panels are designed for PE150 sequencing.

Figure 1: NEBNext Direct Workflow


Next Direct® Custom Ready Panels |

 
Figure 2: NEBNext Direct Custom Ready Panels demonstrate optimum performance across a wide range of panel sizes.


Next Direct® Custom Ready Panels |

Key target enrichment metrics demonstrate consistent performance across a range of panel sizes. 100 ng of DNA was tested against panels of 1, 10, 25, 50 and 100 genes, and sequenced using Illumina paired-end 150 bp sequencing. Larger panels included all genes present in smaller panels.
Figure 3: NEBNext Direct Custom Ready Panels demonstrate retention of target behavior across panel sizes.


Next Direct® Custom Ready Panels |

IGV image of coverage profile for 4 BRAF exons included in panels of 1, 10, 25, 50 and 100 genes, demonstrate consistent target behavior with the addition of gene targets. 100 ng of DNA was used as input for NEBNext enrichment using the 5 panels, including the BRAF gene. Libraries were sequenced on an Illumina 2 x 150 basepair sequencing.
Figure 4: Sensitivity in detection of variants across panel size and DNA input amount.


Next Direct® Custom Ready Panels |

24 HapMap samples were blended to create a range of variant allele frequencies (VAF) down to 2%. 25, 50, 100, 200 and 500 ng of this blended DNA was enriched using NEBNext Direct Custom Ready Panels of 1, 10, 25, 50, and 100 genes. Larger panels were inclusive of the genes in smaller panels. Resulting libraries were sequenced using 2 x 150 bp Illumina sequencing and variants were called using Mutect and Vardict variant calling algorithms.
Figure 5: NEBNext Direct Nicking Enzyme produces consistent fragmentation across DNA input quantities.


Next Direct® Custom Ready Panels |

10 ng, 100 ng, and 1 µg of Control DNA was used as input for fragmentation using the NEBNext Direct Nicking Enzyme. Representative electropherograms show fragment sizes compatible with NEBNext Direct enrichment across DNA input amounts.
Figure 6: NEBNext Direct Nicking Enzyme performance vs. mechanical shearing


Next Direct® Custom Ready Panels |

100 ng of Control DNA was used as input to fragmentation using the NEBNext Direct Nicking Enzyme and Covaris® Shearing. Fragmented DNA was enriched using the 37 kb NEBNext Direct Cancer HotSpot Panel, and sequenced to a mean depth of 3 million reads using Illumina 2 x 75 bp sequencing.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C) Concentration
NEBNext Direct® Bead Prep Buffer 4
NEBNext Direct® dA-Tailing Buffer 4
NEBNext Direct® Adaptor Ligation Buffer 4
NEBNext Direct® Cleaving Buffer 4
NEBNext Direct® Bead Wash 1 25
NEBNext Direct® Bead Wash 2 4
NEBNext Direct® 3´ Adaptor -20
NEBNext Direct® 5´ UMI Adaptor -20
NEBNext Direct® dA-Tailing Enzyme -20
NEBNext Direct® Ligase -20
NEBNext Direct® 5´ Blunting Enzyme Mix -20
NEBNext Direct® Cleaving Enzyme Mix -20
NEBNext Direct® Q5 Master Mix -20
NEBNext Direct® Index Primer Mix D01 (S and L) -20 10 µM
NEBNext Direct® Index Primer Mix D02 (S and L) -20 10 µM
NEBNext Direct® Index Primer Mix D03 (S and L) -20 10 µM
NEBNext Direct® Index Primer Mix D04 (S and L) -20 10 µM
NEBNext Direct® Index Primer Mix D05 (S and L) -20 10 µM
NEBNext Direct® Index Primer Mix D07 (S and L) -20 10 µM
NEBNext Direct® Index Primer Mix D08 (S and L) -20 10 µM
NEBNext Direct® Index Primer Mix D09 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D10 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D11 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D12 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D13 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D14 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D15 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D16 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D17 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D18 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D19 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D20 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D21 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D22 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D23 (L) -20 10 µM
NEBNext Direct® Index Primer Mix D24 (L) -20 10 µM
NEBNext Direct® Index Primer Mix Plate (X) -20 10 µM
NEBNext Sample Purification Beads 4
NEBNext Direct® Streptavidin Beads 4
NEBNext Direct® Hybridization Additive -20
NEBNext Direct® 3´ Blunting Buffer 4
NEBNext Direct® 5´ Blunting Buffer 4
NEBNext Direct Hybridization Wash (HW) 4
NEBNext Direct® Hybridization Buffer 4
NEBNext Direct® 3´ Blunting Enzyme Mix -20
NEBNext Direct® DNA Nicking Buffer -20
NEBNext Direct® DNA Nicking Enzyme -20
NEBNext Direct® Stop Solution -20
NEBNext Direct® Custom Ready Baits
产品类别:
Discontinued Products

  • 特性和用法

    需要但不提供的材料

    1X TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
    Molecular grade ethanol
    Molecular grade water
    0.2 ml PCR strip tubes or plates
    Eppendorf® DNA LoBind® 2 ml tubes (VWR, cat#: 80077-234)
    Additional microcentrifuge or conical tubes to prepare master mixes
    96-well plate magnet or PCR tube magnet
    Microcentrifuge tube magnet
    Agilent High Sensitivity DNA Kit (Agilent, cat#: 5067-4626) or similar

  • 优势和特性

    应用特性

    Each NEBNext Direct Custom Ready Panel is designed to enrich for the complete exonic content of genes selected from the Custom Ready portfolio for next-generation sequencing on the Illumina platform (Illumina, Inc). This kit contains the oligonucleotides, beads, enzymes and buffers required to convert the desired fragments into a sequence-ready library containing both an 8 bp sample index in the Illumina i7 index location and a 12 bp unique molecular identifier (UMI) in the Illumina i5 index location. The NEBNext Direct enrichment method can be performed within one to two days and is easily automated.

    Lot Control: Each set of E6635 reagents is functionally validated together through construction and sequencing of a target enriched DNA library on an Illumina sequencing platform using the NEBNext Direct Cancer HotSpot Baits.

    For 100 ng of human genomic DNA input and an average coverage of 100X, 100% of the targets are covered using the NEBNext Direct Cancer HotSpot Baits. For each Custom Ready Panel, reagents are functionally validated together through construction and sequencing of a target enriched DNA library on an Illumina sequencing platform. Test results may vary based on the selection of targets tested.

操作说明、说明书 & 用法

  • 操作说明
    1. Protocol for NEBNext Direct Custom Ready Panels (NEB# E6631)
    2. Guidelines for Setting up PCR Reactions (E6631X only)

  • 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。

    • manualE6631

  • 使用指南
    • Index Pooling Guidelines for E6631
    • Sample Sheet for E6631L with the index sequences that can be used in the Illumina® experiment Manager v4
    • Sample Sheet for E6631L with the index sequences that can be used in the Illumina® experiment Manager v5
    • Sample Sheet for E6631S with the index sequences that can be used in the Illumina® experiment Manager v5
    • Sample Sheet for E6631S with the index sequences that can be used in the Illumina® experiment Manager v4 
    • Sample Sheet for E6631X with the index sequences that can be used in the Illumina® experiment Manager v4
    • Sample Sheet for E6631X with the index sequences that can be used in the Illumina® experiment Manager v5

FAQs & 问题解决指南

  • FAQs
    1. What size should I fragment my DNA to?
    2. If my FFPE DNA is fragmented, should I do a shearing step?
    3. Can I repair my FFPE DNA using the NEBNext FFPE DNA Repair Mix (NEB #M6630)?
    4. How can I warm Bead Wash 1 (BW1) to dissolve any precipitate?
    5. If I have adaptor dimer in my finished library, can I perform another bead cleanup to remove it?
    6. Where can I find the bed files for my panel?
    7. Are there sample sheets available to ensure sequencer runs are configured appropriately?
    8. Can NEBNext Direct be applied for genotyping applications?
    9. How many times can the -20°C reagents be frozen and thawed?
    10. How many times can the 4°C reagents be brought to temperature?
    11. My 4°C box accidentally froze, can I still use it?
    12. I accidentally stored the Sample Purification Beads and Bead Wash 1 (BW1) at 4°C. Can I still use them?
    13. What is the shelf life of this product?
    14. What method do you recommend for extracting genomic DNA and FFPE DNA before use with the NEBNext Direct Panel?
    15. Can shearing be done using Covaris®?
    16. What is the recommended method for quantitating my input DNA?
    17. If my DNA input material amount is high, should I reduce the number of PCR cycles?
    18. If my input amount is low, should I dilute the adaptors?
    19. How much starting material do I need to use when preparing libraries using the NEBNext Direct Panel?
    20. Which magnets (2 ml and 200 μl sizes) do you recommend?
    21. Is it ok to leave bead separations for longer than 15 seconds?
    22. I used Bead Wash 1 (BW1) when I should have used Bead Wash 2 (BW2) during the Post-reaction Wash. Can I do another wash with Bead Wash 2 (BW2) to save my sample?
    23. I used Bead Wash 2 (BW2) when I should have used Bead Wash 1 (BW1) during the Post-reaction Wash. What should I do?
    24. Does incomplete removal of Bead Wash 1 prior to adding Bead Wash 2 inhibit the next step?
    25. Can I use any Q5® formulation with this kit?
    26. How many/ few samples can I pool together for my sequencing run?
    27. Are the indexes in E7000S/ E6627S/E6631S (D01-D08) the same sequences as the first 8 indexes (D01-D08) in NEB #E7000L/ E6627L/E6631L?

引用 & 技术文献

  • 产品手册
    • Ordering Guide

  • 引用文献

    NEBNext Product Citations Search Tool

    Use this tool to find citations related to NEBNext products. Search by product, keyword or instrument name – use the filters to select specific product areas.

  • 专题文章
    • Overcoming the challenges of applying target enrichment for translational research

质控、安全 & 法规

  • 质控分析
    每一新批次的 NEB 产品都要经过质控，以满足指定的质量标准，对特定产品的质量标准和每一批次的检测数据可以通过产品说明表格、COA、产品信息卡或者产品手册进行查阅或下载。关于 NEB 产品质控的详细信息，可从此处查阅。

  • 产品说明与变更通知

    产品说明与变更通知

    产品说明表包含产品的储存温度、有效期和规格，这些文件的命名规则如下：[货号]_[规格]_[版本]

    • E6631L_v1
    • E6631S_v1
    • E6631X_v1

  • SDS
    以下 SDS 文件可以帮助您安全地使用该产品

    • NEBNext Direct® Custom Ready Panels