Product Class:

NEBNext Direct® Custom Ready Panels

Use our NEBNext Direct® Custom Ready Panel Request Form to order. 
 
    Item# Description List Price Web Price Qty
    E6631 NEBNext Direct Custom Ready Panels $0.00
    $0.00
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    *On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

    Product Introduction

    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 Molecule Index (UMI)
    • Generate results in one day with our automation-friendly workflow

    Product Information

    Description

    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




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




    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.




    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.




    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.




    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




    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.

    Each set of reagents is functionally validated together through construction and sequencing of a transcriptome library and sequenced on a

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    NEBNext Direct® Bead Prep Buffer 4 0 %
    NEBNext Direct® dA-Tailing Buffer 4 0 %
    NEBNext Direct® Adaptor Ligation Buffer 4 0 %
    NEBNext Direct® Cleaving Buffer 4 0 %
    NEBNext Direct® Bead Wash 1 25 0 %
    NEBNext Direct® Bead Wash 2 4 0 %
    NEBNext Direct® 3´ Adaptor -20 0 %
    NEBNext Direct® 5´ UMI Adaptor -20 0 %
    NEBNext Direct® dA-Tailing Enzyme -20 0 %
    NEBNext Direct® Ligase -20 0 %
    NEBNext Direct® 5´ Blunting Enzyme Mix -20 0 %
    NEBNext Direct® Cleaving Enzyme Mix -20 0 %
    NEBNext Direct® Q5 Master Mix -20 0 %
    NEBNext Index Primer Mix D01-D08 (E7020-E7027) -20 10 μM
    NEBNext Direct® Index Primer Mix D02 -20 10 μM
    NEBNext Direct® Index Primer Mix D03 -20 10 μM
    NEBNext Direct® Index Primer Mix D04 -20 10 μM
    NEBNext Direct® Index Primer Mix D05 -20 10 μM
    NEBNext Direct® Index Primer Mix D07 -20 10 μM
    NEBNext Direct® Index Primer Mix D08 -20 10 μM
    NEBNext Direct® Index Primer Mix D09 -20 10 μM
    NEBNext Direct® Index Primer Mix D10 -20 10 μM
    NEBNext Direct® Index Primer Mix D11 -20 10 μM
    NEBNext Direct® Index Primer Mix D12 -20 10 μM
    NEBNext Direct® Index Primer Mix D13 -20 10 μM
    NEBNext Direct® Index Primer Mix D14 -20 10 μM
    NEBNext Direct® Index Primer Mix D15 -20 10 μM
    NEBNext Direct® Index Primer Mix D16 -20 10 μM
    NEBNext Direct® Index Primer Mix D17 -20 10 μM
    NEBNext Direct® Index Primer Mix D18 -20 10 μM
    NEBNext Direct® Index Primer Mix D19 -20 10 μM
    NEBNext Direct® Index Primer Mix D20 -20 10 μM
    NEBNext Direct® Index Primer Mix D21 -20 10 μM
    NEBNext Direct® Index Primer Mix D22 -20 10 μM
    NEBNext Direct® Index Primer Mix D23 -20 10 μM
    NEBNext Direct® Index Primer Mix D24 -20 0 %
    NEBNext Direct® Index Primer Mix Plate (NEB #E7000X) -20 0 %
    NEBNext Sample Purification Beads 4 0 %
    NEBNext Direct® Streptavidin Beads 4 0 %
    NEBNext Direct® Hybridization Additive -20 0 %
    NEBNext Direct® 3´ Blunting Buffer 4 0 %
    NEBNext Direct® 5´ Blunting Buffer 4 0 %
    NEBNext Direct Hybridization Wash (HW) 4 0 %
    NEBNext Direct Hybridization Buffer 4 0 %
    NEBNext Direct® 3´ Blunting Enzyme Mix -20 0 %
    NEBNEXT DIRECT™ DNA NICKING BUFFER 0 %
    NEBNext Direct® DNA Nicking Enzyme 0 %
    NEBNext Direct® Stop Solution 0 %
    NEBNext Direct Custom Ready Baits 0 %
    Product Categories:
    Targeted Enrichment Products,
    Sample Prep for NGS & Target Enrichment Products
    Applications:
    DNA Library Preparation,
    Sample Prep for NGS & Target Enrichment

    Advantages and Features

    Application Features

    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.

    Properties and Usage

    Materials Required but not Supplied

    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

    Storage Temperature

    Multi-Temperature°C

    Faqs & Tech Tips

    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?

    Protocols & Manuals

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

    Protocols

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

    Other Tools & Resources

    Featured Articles

    • Overcoming the challenges of applying target enrichment for translational research

    Usage & Guidelines

    Quality & Safety

    Quality Assurance Statement

    Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

    Specifications

    The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

    Safety DataSheets

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    NEBNext Direct® Bead Prep Buffer
    NEBNext Direct® dA-Tailing Buffer
    NEBNext Direct® Adaptor Ligation Buffer
    NEBNext Direct® Cleaving Buffer
    NEBNext Direct® Bead Wash 1
    NEBNext Direct® Bead Wash 2
    NEBNext Direct® 3´ Adaptor
    NEBNext Direct® 5´ UMI Adaptor
    NEBNext Direct® dA-Tailing Enzyme
    NEBNext Direct® Ligase
    NEBNext Direct® 5´ Blunting Enzyme Mix
    NEBNext Direct® Cleaving Enzyme Mix
    NEBNext Direct® Q5 Master Mix
    NEBNext Index Primer Mix D01-D08 (E7020-E7027)
    NEBNext Index Primer Mix D01-D08 (E7020-E7027)
    NEBNext Index Primer Mix D01-D08 (E7020-E7027)
    NEBNext Direct® Index Primer Mix Plate (NEB #E7000X)
    NEBNext Direct® Streptavidin Beads
    NEBNext Direct® Hybridization Additive
    NEBNext Direct® 3´ Blunting Buffer
    NEBNext Direct® 3´ Blunting Buffer
    NEBNext Direct® 5´ Blunting Buffer
    NEBNext Direct® 5´ Blunting Buffer
    NEBNext Direct Hybridization Wash (HW)
    NEBNext Direct Hybridization Buffer
    NEBNext Direct® 3´ Blunting Enzyme Mix
    NEBNext Direct® 3´ Blunting Enzyme Mix