Catalog # E6627 was discontinued on December 31, 2020
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
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.
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 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 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 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
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.
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
Advantages and Features
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 custom@ neb.com for further information.
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Specifications & Change Notifications
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The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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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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