Product Class: Kit

NEBNext Direct® BRCA1/BRCA2 Panel

This product was discontinued on 12/31/20. In place of E6627, the content can be ordered as NEBNext Direct Custom Ready Panels.

  • Catalog # E6627 was discontinued on December 31, 2020

Product Introduction

NEBNext Direct employs a unique hybridization-based enrichment workflow that hybridizes baits directly to genomic DNA, without the need for upfront library preparation. The BRCA1/BRCA2 panel demonstrates extremely high specificity and unmatched coverage uniformity across a wide range of DNA inputs, allowing highly sensitive calling of germline and somatic variants while maximizing sequencer efficiency. 

  • Generate full (100%) coverage of all protein coding regions in BRCA1 and BRCA2 genes
  • Obtain highly uniform sequencing across exon targets- 100% of base pairs in panel have coverage greater than 20% of the mean target coverage
  • Maximize efficiency with a 1-day workflow that combines highly specific enrichment with library preparation
  • Produce high depths of target coverage across a wide range of DNA input amounts for germline and somatic variant calling
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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 BRCA1/BRCA2 Panel is designed to enrich for the complete exonic content of the BRCA1 and BRCA2 genes. 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 sequencing.

Figure 1. NEBNext Direct Workflow.
 fast hybridization workflow

The NEBNext Direct BRCA1/BRCA2 Panel delivers highly efficient enrichment of BRCA1 and BRCA2 coding regions with a high percentage of reads mapping to targets
NEBNext Direct BRCA1/BRCA2 Panel
This histogram shows the percent of reads aligned to the human genome, and the percent of reads mapped to the targets included in the BRCA1/BRCA2 Panel across different input DNA amounts. 10 ng, 100 ng and 1 µg of purified genomic DNA was enriched using the NEBNext Direct BRCA1/BRCA2 Panel. Sequencing reads were generated on an llumina® MiSeq® with 2x75 bp reads, 8 bp Sample ID and 12 bp unique molecular identifier. Sequencing read alignments were performed with BWA-MEM, and PCR duplicates were filtered using the UMIs.


The NEBNext Direct BRCA1/BRCA2 Panel produces highly uniform coverage across target regions, maximizing sequencer efficiency
specificity of BRCA testing histogram
This histogram shows the normalized mean coverage across each of the 49 exon targets of the BRCA1 and BRCA2 genes. 50 ng of DNA was enriched using the 17 kb NEBNext Direct BRCA1/BRCA2 Panel. Sequencing reads were generated on an Illumina MiSeq with 2x75 bp reads, an 8 bp sample ID and 12 bp unique molecular identifier. Sequencing read alignments were performed with BWA-MEM, and PCR duplicates were filtered using the UMIs.


The NEBNext Direct BRCA1/BRCA2 Panel can be used to provide depths of sequencing compatible with both germline and somatic variant calling
BRCA Converge Uniformity Histogram
This figure demonstrates de-duplicated coverage of BRCA1 and BRCA2 genes as a function of sequencing depth across a range of input DNA amounts. Purified genomic DNA was enriched using the NEBNext Direct BRCA1/BRCA2 Panel. Sequencing reads were generated on an Illumina MiSeq with 2x75 bp reads, the 8 bp sample ID and 12 bp unique molecular identifier. Sequencing read alignments were performed with BWA-MEM, and PCR duplicates were filtered using the UMIs.


The NEBNext Direct BRCA1/BRCA2 Panel produces even depths of coverage across target regions
coverage of BRCA1 and BRCA2 genes graph

Integrative Genome Viewer (IGV) plot showing the read coverage across exon 1 of the BRCA1 gene. 50 ng of DNA was enriched using the 17 kb NEBNext Direct BRCA1/BRCA2 Panel. Sequencing reads were generated on an Illumina MiSeq with 2x75 bp reads, an 8 bp sample ID, and 12 bp unique molecular identifier. Sequencing read alignments were performed with BWA-MEM, and PCR duplicates were filtered using the UMIs.

NEBNext Direct® BRCA1/BRCA2 Resources:

 

This product is related to the following categories:
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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

Application Features

The NEBNext Direct BRCA1/BRCA2 Panel for Illumina® is designed to enrich for complete exonic content for BRCA1 and BRCA2 genes 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 containing both sample indexes and unique molecular identifiers (UMI). The NEBNext Direct enrichment method can be performed within one day and is easily automated. Each kit component must pass rigorous quality control standards and is lot controlled both individually and as a set of reagents.

Lot Control: The lots provided in the NEBNext Direct BRCA1/BRCA2 Panel are managed separately and qualified by additional functional validation. Individual reagents undergo standard enzyme activity and quality control assays.

Functional Validation: Each set of reagents is functionally validated together through construction and sequencing of a target enriched DNA library on an Illumina sequencing platform. For 100 ng of NA19240 DNA input and an average coverage of 100X, 100% of the targets are covered.

Need a Bulk Order?
For larger volume requirements, customized and bulk packaging is available by purchasing through the Custom Solutions Team at NEB. Please contact the Customized Solutions for further information.

FAQs & Troubleshooting

FAQs

  1. Can NEBNext Direct be applied for genotyping applications?
  2. What size should I fragment my DNA to?
  3. Can I repair my FFPE DNA using the NEBNext FFPE DNA Repair Mix (NEB #M6630)?
  4. How can I warm Bead Wash 1 (BW1) to dissolve any precipitate?
  5. If I have adaptor dimer in my finished library, can I perform another bead cleanup to remove it?
  6. How many times can the -20°C reagents be frozen and thawed?
  7. How many times can the 4°C reagents be brought to room temperature?
  8. My 4°C box accidentally froze, can I still use it?
  9. I accidentally stored the Sample Purification Beads and Bead Wash 1 (BW1) at 4°C. Can I still use them?
  10. What method do you recommend for extracting genomic DNA and FFPE DNA before use with the NEBNext Direct Panel? 
  11. Can shearing be done using Covaris®?
  12. What is the recommended method for quantitating my input DNA?
  13. If my DNA input material amount is high, should I reduce the number of PCR cycles?
  14. If my input amount is low, should I dilute the adaptors?
  15. What type and how much starting material do I need to use when preparing libraries using the NEBNext Direct Panel?
  16. Which magnets (2 ml and 200 µl sizes) do you recommend?
  17. Is it ok to leave bead separations for longer than 15 seconds?
  18. 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?
  19. I used Bead Wash 2 (BW2) when I should have used Bead Wash 1 (BW1) during the Post-reaction Wash. What should I do?
  20. Does incomplete removal of Bead Wash 1 prior to adding Bead Wash 2 inhibit the next step?
  21. Can I use any Q5® formulation with this kit?
  22. How many sequencing reads per sample do you recommend using with the NEBNext Direct BRCA1/BRCA2 Panel?
  23. How many/ few samples can I pool together for my sequencing run?
  24. Are the indexes in E7000S/ E6627S/E6631S (D01-D08) the same sequences as the first 8 indexes (D01-D08) in NEB #E7000L/ E6627L/E6631L?
  25. If my FFPE DNA is fragmented, should I do a shearing step?

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 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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