Technology

The NEBNext Direct® technology uses a novel approach to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes, without sacrificing specificity. The approach rapidly hybridizes both strands of genomic DNA with biotinylated baits, captures the targets on streptavidin beads, enzymatically removes of off-target sequence, and directly converts captured molecules into Illumina-ready libraries (Figure 1).

The sequencing reads have highly defined beginning and end points and produce uniform coverage across a given target. Unlike alternative hybridization methods, NEBNext Direct® does not require upfront library preparation. Instead, target molecules acquire either a single or dual index sample ID and a 12bp Unique Molecule Index (UMI) as part of the targeting protocol. The UMI tags each individual molecule prior to the final PCR amplification and enables identification of PCR duplicates. All of these steps are accomplished in a 6.5-hour protocol that produces sequence-ready libraries specific to the gene content in the panel from purified genomic DNA.

Advantages

  • Generate a higher percentage of your sequencing reads aligning to your targets
  • Obtain uniform sequencing of ALL targets, regardless of GC content
  • Save time with a streamlined workflow that couples enrichment and 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
  • On-bead sample preparation is ideal for automated workflows

More information is available on the New England Biolab’s website:
https://www.neb.com/nebnext-direct/nebnext-direct-for-target-enrichment





Figure 1. NEBNext Direct® combines capture with library preparation in a single fast, hybridization-based workflow.




Performance

Mean target depth, after removal of PCR duplicates, obtained across a variety of panel sizes and DNA input amounts with 16 hr hybridizaiton and increasing number of sequencing reads.