NEBNext Direct® Cancer HotSpot Panel
Challenges and Opportunities for Next Generation Sequencing Target Enrichment
NEBNext Direct® Target Enrichment
NEBNext Direct® Workflow
NEB TV Ep. 15 – Applications of Restriction Enzymes
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.
- 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.
Table 1. 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.
Figure 3. The NEBNext Direct Cancer HotSpot Panel delivers a high percentage of sequence reads mapping to targets, even with challenging sample types.
Figure 4. The NEBNext Direct Cancer HotSpot Panel displays high uniformity of coverage across targets.
Figure 5. The NEBNext Direct Cancer HotSpot Panel offers minimized bias across sequence content.
ILLUMINA® and MISEQ® are registered trademarks of Illumina®, Inc.
Lot ControlThe 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
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® FFPE Phosphorylation Enzyme -20 NEBNext Direct® FFPE Phosphorylation Buffer -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
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