This is a brief introduction to the NEBNext Direct technology for targeted enrichment of genomic regions of interest prior to next-generation sequencing, and an introduction to the NEBNext Direct Cancer HotSpot Panel, a focused panel comprised of 190 targets from 50 cancer related genes.
NEBNext Direct uses a novel approach for target enrichment and is capable of enriching a wide range of gene targets ranging from single gene tests to comprehensive panels comprised of several hundred genes. The technology offers high specificity and uniformity across targets, while enabling high-sensitivity calling of nucleic acid variants including single nucleotide polymorphisms and indels.
A simple 1-day workflow that combines target enrichment and library preparation was designed with automation in mind. The NEBNext Direct approach offers significant advantages over both traditional in-solution hybridization and multiplex PCR protocols, and accommodates multiple sample types and input materials, including FFPE tissue and cell-free DNA.
The NEBNext Direct protocol begins with mechanically fragmented DNA and hybridizes a short biotinylated probe to each strand of the denatured target molecule. These bound fragments are then physically separated using magnetic streptavidin beads. Off target sequence is enzymatically removed through 3’ blunting of the DNA molecule, to which the 3’ adapter is ligated. 5’ blunting of the DNA extends the probe across the full length of the target molecule, creating a variable 5’ end depending on the specific size of the sheared fragment. A 5’ adapter containing a 12 base pair unique molecular identifier is then ligated to the 5’ end of the fragment. The 3’ adaptor is cleaved, and PCR amplification is performed to release the captured strands from the probe/bead complex, as well as to add the sample index.
The result is a sequence-ready library. The protocol takes less than one day to complete, and the combination of “on-bead” sample preparation and the elimination of user interventions render the workflow highly amenable to automation.
NEBNext Direct incorporates two types of indexes during the workflow. The sample ID is added during PCR in the i7 index position and enables samples to be pooled together prior to sequencing in order to maximize efficiency of the sequencing run. There are currently 120 unique sample ID’s available.
The Unique Molecule ID is added to the i5 position. This ID contains a randomized 12bp sequence and is incorporated in the i5 index position. Incorporation of this randomized sequence is independent for each fragment in the sample, and is used to identify duplicate molecules that can be created during PCR. Bioinformatic utilization of these indexes can be used to reduce false-positive variants that can be introduced through sample handing and preparation processes.
It is important to note that NEBNext Direct captures both strands of DNA from each molecule. The 3’ end of each molecule is defined by the enzymatic removal of the 3’ off target sequence, and the variable 5’ end is defined by random fragmentation of the initial target molecule. The variable nature of the 5’ end can also be used to de-duplicate reads, and provide a more quantitative assessment of variant frequencies in the original sample.
The NEBNext Direct technology has proven to maintain high specificity across a wide range of target sizes and types demonstrating high percent on target reads in assays ranging from 2 genes to 133 genes including exons, promoters, non-coding regions, and CNV controls. This flexibility allows laboratories to maintain a single workflow for a wide range of assays.
Moving on, we will describe the NEBNext Direct Cancer HotSpot Panel.
The NEBNext Direct Cancer HotSpot panel is a defined content panel that utilizes the NEBNext Direct approach to perform highly specific enrichment from 190 common cancer targets across 50 genes. The panel provides full exon coverage of targets, with a total target territory of 37 kilobases, covering over 18,000 features from the Catalogue of Somatic Mutations in Cancer database, or COSMIC.
Careful optimization of the panel results in extremely uniform coverage across targets, reducing overall sequencing costs, and eliminating any dropouts. The panel’s targets have been optimized for Illumina 2 x 75 bp sequencing.
The NEBNext Direct Cancer HotSpot Panel contains all of the reagents needed to go from extracted genomic DNA to a sequence-ready library. The kits include all of the required enzymes and buffers, oligonucleotides including hybridization probes, adaptors, and primers, as well as necessary streptavidin and sample purification magnetic beads.
The kits are available in 3 sizes, an 8 reaction kit, a 24 reaction kit, and a 96 reaction kit. There are a total of 120 sample indexes available allowing for optimal pooling to maximize sequencer efficiency.
The NEBNext Direct Cancer HotSpot Panel produces high specificity for targeted regions as demonstrated in the percentage of reads mapping to targets. This high specificity reduces overall sequencing costs, and is shown to persist across challenging sample types as seen here in libraries derived from FFPE tissue and cell-free DNA.
Individually synthesized probes allow for re-balancing of specific probes to ensure even coverage across targets. Careful consideration has been put in place to ensure a high degree of coverage uniformity across targeted regions, with 100% of targets covered to greater than 25% of the mean target coverage, and 90% of targets covered to greater than 50% of the mean target coverage. This uniformity significantly reduces the overall amount of sequencing required to achieve the minimum depth of coverage required for somatic variant calling.
The NEBNext Cancer HotSpot Panel has been optimized to work well across a wide range of gene content as seen here as we look at normalized coverage across the range of GC content represented by the Panel, again allowing the reduction in overall sequencing in order to meet coverage requirements.
In summary, the NEBNext Direct technology presents a tractable approach that combines the specificity and ease of multiplexed amplicon-based panels with the content scale more typical of hybridization based panels. Incorporated Unique Molecule IDs and variable insert sizes enable de-duplication of reads and improve sensitivity for variant calling. NEBNext Direct provides the specificity, uniformity and sensitivity capable of detecting low-frequency variants across a range of sample types.
For more information on NEBNext Direct or the NEBNextDirect Cancer HotSpot Panel, or to inquire about testing the technology in your hands, please email [email protected], or visit us online at nebnextdirect.com
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