NEBNext Direct® Cancer HotSpot Panel

Description







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® FFPE Phosphorylation Enzyme-20
NEBNext Direct® FFPE Phosphorylation Buffer-20
NEBNext Direct® Hybridization Additive-20
NEBNext Direct® dA-Tailing Enzyme-20
NEBNext Direct® 3´ Adaptor-20
NEBNext Direct® Ligase-20
NEBNext Direct® 5´ Blunting Enzyme Mix-20
NEBNext Direct® 5´ UMI Adaptor-20
NEBNext Direct® Cleaving Enzyme Mix-20
NEBNext Direct® Q5 Master Mix-20
NEBNext Index Primer Mix D01-D08 (E7020-E7027)-20
NEBNext Direct® Bead Wash 125
NEBNext Direct® Streptavidin Beads4
NEBNext Direct® Hybridization Wash (HW)4
NEBNext Direct® Bead Prep Buffer4
NEBNext Direct® dA-Tailing Buffer4
NEBNext Direct® Adaptor Ligation Buffer4
NEBNext Direct® Cleaving Buffer4
NEBNext Direct® Bead Wash 24
NEBNext Direct® 3´ Blunting Enzyme Mix-20
NEBNext Direct® 5´ Blunting Buffer4
NEBNext Direct Cancer HotSpot Baits-20
NEBNext Sample Purification Beads4
NEBNext Direct Hybridization Buffer4

Advantages and Features

Features

  • 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

Properties and Usage

Materials Required but not Supplied

  • Covaris® microTubes or plate
  • 1X TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
  • Molecular grade ethanol
  • Molecular grade water
  • 96-well PCR plates (or PCR strip tubes)
  • 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)

Required Equipment:

  • Covaris Focused-Ultrasonicator
  • Thermocycler programmable to 100 μl
  • Agilent Bioanalyzer® or similar instrument

FAQs

  1. Where can I find the detailed FAQs for the NEBNext Direct™ Cancer HotSpot Panel (NEB #E7000)?

Protocols

  1. Index Pooling Guidelines (NEB #E7000)
  2. Guidelines for Setting Up PCR Reactions (NEB #E7000X only)
  3. Protocol for NEBNext Direct® Cancer HotSpot Panel (NEB #E7000)
  4. Guidelines for Running Samples on the Illumina MiSeq (E7000)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

Usage Guidelines & Tips

Safety Data Sheet

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