NEBNext Direct® BRCA1/BRCA2 Panel

Description

Sample

 
 
The NEBNext Direct BRCA1/BRCA2 Panel enriches the complete exon content of BRCA1 and BRCA2 genes for next-generation se­quencing analysis. 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.

Benefits:

  • Generate full (100%) coverage of all protein coding regions in BRCA1 and BRCA2 genes
  • Obtain highly uniform sequencing across exon targets- 100% of basepairs 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.

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
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.

Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBNext Direct® Hybridization Additive-20
NEBNext Direct® 3´ Blunting Enzyme Mix-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® FFPE Phosphorylation Enzyme-20
NEBNext Direct® Bead Wash 125
NEBNext Sample Purification Beads25
NEBNext Direct® Streptavidin Beads4
NEBNext Direct® Hybridization Wash (HW)4
NEBNext Direct® Bead Prep Buffer4
NEBNext Direct® Hybridization Buffer4
NEBNext Direct® dA-Tailing Buffer4
NEBNext Direct® Adaptor Ligation Buffer4
NEBNext Direct® 5´ Blunting Buffer4
NEBNext Direct® Cleaving Buffer4
NEBNext Direct® Bead Wash 24
NEBNext Direct® Q5 Master Mix-20
NEBNext Direct BRCA1/BRCA2 Baits-20

Advantages and Features

Applications

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. 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.

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® BRCA1/2 Panel?

Protocols

  1. Protocol for NEBNext Direct BRCA1/BRCA2 Panel (E6627)
  2. Guidelines for Setting up PCR Reactions (E6627)
  3. Index Pooling Guidelines (E6627)
  4. Guidelines for Running Samples on the Illumina MiSeq (E6627)

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

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]

Safety Data Sheet

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