NEBNext Direct® Cancer HotSpot Panel

Please note: product formulation and protocols have been updated. Please download manual for details.

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    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), fragmented DNA is rapidly hybridized to biotinylated oligonucleotide baits that define the 3´ end of each target of interest. The bait-target hybrids are bound to streptavidin beads and any 3´ off target sequence is removed enzymatically. This combination of a short hybridization time with the enzymatic removal of 3´ off target sequence enables greater sequencing efficiency relative to conventional hybridization-based enrichment methods. The trimmed targets are then converted into Illumina-compatible libraries that include unique molecular identifiers (UMI) and a sample barcode. Sequence-ready libraries are generated within one day. The procedure is compatible with most automated liquid handling instruments.

    The NEBNext Direct HotSpot Cancer Panel contains baits that capture both strands of DNA across 190 common cancer targets from 50 genes, encompassing approximately 40 kb of sequence and including over 18,000 COSMIC features (Table 1). The panel is designed to generate targets of roughly 150 bp, compatible with PE75 Illumina sequencing.

    Advantages
    • Generate a higher percentage of your sequencing reads aligning to your targets
    • Eliminate the need to over-sequence, reducing cost per sample
    • Obtain uniform sequencing of all targets, regardless of GC content
    • Save time with a 1-day workflow that combines enrichment with library preparation
    • Generate high quality libraries with limited input amounts and degraded DNA samples, including FFPE and ctDNA
    • Distinguish molecular duplicates, reducing false positive variants and improving sensitivity


    Figure 1. NEBNext Direct employs a fast hybridization-based workflow that combines capture with library preparation.

    fast hybridization workflow


    Table 1. Targets include regions from the following cancer-related genes:

    Targets include regions from the following cancer-related genes


    Figure 2. The NEBNext Direct Cancer HotSpot Panel demonstrates the ability to accurately detect a range of nucleic acid variants.

    Allele Frequency
    This figure shows the expected versus observed variant allele frequencies (VAF) across the range of well-characterized variants present in a pool of 24 HapMap samples screened against the NEBNext Direct Cancer HotSpot Panel. 100 ng of input DNA was used, samples were sequenced on the Illumina® MiSeq® using 2 x 75 bp sequencing, and standard data analysis and variant calling algorithms were used. We were able to successfully detect 100% of the 168 truth variants present across a range of 2-100% VAF. The high degree of linearity across this broad dynamic range demonstrates the ability of the NEBNext Direct Cancer HotSpot Panel to accurately predict variant allele frequencies across a broad dynamic range.


    Figure 3. The NEBNext Direct Cancer HotSpot Panel delivers a high percentage of sequence reads mapping to targets, even with challenging sample types.

    The NEBNext Direct Cancer HotSpot Panel delivers a high percentage of sequence reads mapping to targets, even with challenging sample types.

    • Graph shows the percentage of aligned sequence reads that map to the targets
    • 100 ng of DNA was used for each library preparation
    • Reads were generated on an Illumina® MiSeq® with 2 x 75 bp reads, 8 bp sample ID, and 12 bp unique molecule ID
    • Alignments were performed with BWA-MEM and PCR duplicates were filtered using the unique molecule IDs

    Figure 4. The NEBNext Direct Cancer HotSpot Panel displays high uniformity of coverage across targets.

     Cancer HotSpot Panel displays high uniformity of coverage across targets.

    • Graph shows the percentage of target bases sequenced to at least 50%, 33%, and 25% of the mean read depth
    • 100 ng of DNA was used for each library preparation
    • Reads were generated on an Illumina MiSeq with 2 x 75 bp reads, 8 bp sample ID, and 12 bp unique molecule ID
    • Alignments were performed with BWA-MEM and PCR duplicates were filtered using the unique molecule IDs


    Figure 5. The NEBNext Direct Cancer HotSpot Panel offers minimized bias across sequence content.

     minimized bias across sequence content

    • Graph shows the normalized depth of coverage of targets of varying GC content
    • 100 ng of DNA was used for each library preparation
    • Reads were generated on an Illumina MiSeq with 2 x 75 bp reads, 8 bp sample ID, and 12 bp unique molecule ID
    • Alignments were performed with BWA-MEM and PCR duplicates were filtered using the unique molecule IDs

    ILLUMINA® and MISEQ® are registered trademarks of Illumina®, Inc.

    Lot Control

    The lots provided are managed separately and qualified by additional functional validation. Individual reagents undergo standard enzyme activity and quality control assays, and also meet stringent criteria in the additional quality controls listed on each individual component page

    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® Hybridization Wash (HW) 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 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 D06 -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
    NEBNext Direct® Index Primer Mix Plate (NEB #E7000X) -20
    NEBNext Sample Purification Beads 4
    NEBNext Direct® Streptavidin Beads 4
    NEBNext Direct® Hybridization Additive -20
    NEBNext Direct® 5´ Blunting Buffer 4
    NEBNext Direct Hybridization Buffer 4
    NEBNext Direct® 3´ Blunting Enzyme Mix -20
    NEBNext Direct Cancer HotSpot Baits -20
    Product Categories:
    Targeted Enrichment
    Applications:
    DNA Library Preparation,
    Sample Preparation for Next Generation Sequencing,
    Illumina Library Preparation
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