Catalog # E6631 was discontinued on December 21, 2021
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.
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.
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
This product is related to the following categories:
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
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.
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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 & Change Notifications
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]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
Licenses
This product is covered by one or more patents.
This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.
For additional information or to inquire about commercial use, please contact busdev@neb.com.
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