Product Class: Kit

NEBNext Direct® Cancer HotSpot Panel

This product was discontinued on 12/31/20. In case there is need for additional supply beyond this date, please contact NEBNextDirect@neb.com.

  • Catalog # E7000 was discontinued on December 31, 2020

Product Introduction

The NEBNext Direct® Cancer HotSpot Panel employs a unique hybridization-based enrichment workflow that hybridizes baits directly to genomic DNA, without the need for upfront library preparation.

  • 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

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Product Information

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), 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 Cancer HotSpot Panel is designed to enrich for DNA fragments across 190 common cancer targets from 50 genes, encompassing approximately 40 kb of sequence and including over 18,000 COSMIC features. This kit contains the oligonucleotides, beads, enzymes and buffers required to convert the desired fragments into a sequence-ready library for next-generation sequencing on the Illumina platform and is designed for PE75 or PE150 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
This product is related to the following categories:
Discontinued (<3 years),

Properties & 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 & Troubleshooting

FAQs

  1. Can NEBNext Direct be applied for genotyping applications?
  2. What size should I fragment my DNA to?
  3. If my FFPE DNA is fragmented, should I do a shearing step?
  4. Can I repair my FFPE DNA using the NEBNext FFPE DNA Repair Mix (NEB #M6630)?
  5. How can I warm Bead Wash 1 (BW1) to dissolve any precipitate?
  6. If I have adaptor dimer in my finished library, can I perform another bead cleanup to remove it?
  7. How many times can the -20°C reagents be frozen and thawed?
  8. How many times can the 4°C reagents be brought to room temperature?
  9. My 4°C box accidentally froze, can I still use it?
  10. I accidentally stored the Sample Purification Beads and Bead Wash 1 (BW1) at 4°C. Can I still use them?
  11. What method do you recommend for extracting genomic DNA and FFPE DNA before use with the NEBNext Direct Panel? 
  12. Can shearing be done using Covaris®?
  13. What is the recommended method for quantitating my input DNA?
  14. If my DNA input material amount is high, should I reduce the number of PCR cycles?
  15. If my input amount is low, should I dilute the adaptors?
  16. What type and how much starting material do I need to use when preparing libraries using the NEBNext Direct Panel?
  17. Which magnets (2 ml and 200 µl sizes) do you recommend?
  18. Is it ok to leave bead separations for longer than 15 seconds?
  19. I used Bead Wash 1 (BW1) when I should have used Bead Wash 2 (BW2) during the Post-reaction Wash. Can I do another wash with Bead Wash 2 (BW2) to save my sample?
  20. I used Bead Wash 2 (BW2) when I should have used Bead Wash 1 (BW1) during the Post-reaction Wash. What should I do?
  21. Does incomplete removal of Bead Wash 1 prior to adding Bead Wash 2 inhibit the next step?
  22. Can I use any Q5® formulation with this kit?
  23. How many sequencing reads per sample do you recommend using with the NEBNext Direct Cancer HotSpot Panel?
  24. How many/ few samples can I pool together for my sequencing run?
  25. Are the indexes in E7000S/ E6627S/E6631S (D01-D08) the same sequences as the first 8 indexes (D01-D08) in NEB #E7000L/ E6627L/E6631L?

Quality, Safety & Legal

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]

Legal and Disclaimers

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 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 for use only in: (a) standard (not real-time) PCR in the research field only, but not real time PCR or digital PCR; (b) real-time PCR for use as a library preparation quantitation tool in Next Generation Sequencing workflows; (c) any in-vitro diagnostics applications, except for applications using real-time PCR or digital PCR; and (d) any non-PCR applications in DNA sequencing, isothermal amplification, and the production of synthetic DNA.

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