NEBNext Direct® Workflow

View our tutorial for help visualizing NEBNext Direct’s unique and enabling workflow.

Script

NEBNext Direct is a novel method for highly-specific enrichment of gene targets prior to next generation sequencing.

1. The workflow begins with DNA that has been randomly fragmented to an average size of 200 base pairs and denatured.

2. A biotinylated bait of roughly 65 nucleotides is hybridized in solution for 90 minutes. Both strands of DNA are individually targeted with separate baits in solution.

3. The captured fragments are then bound to streptavidin beads, and magnetically separated from the mixture. The captured fragments remain bound to the magnetic streptavidin beads and is converted into a sequence-ready library.

4.The 3-prime, off-target sequence is enzymatically removed, creating a blunt end.

5. The 3-prime end is adenylated, and the adaptor is ligated.

6. In the next step, 5-prime blunting, the 3-prime end of the hybridized bait is extended across the length of each molecule. This extension produces the complementary strand to the target molecule, and results in double-stranded DNA that is ready for ligation of the 5-prime adaptor.

7. The 5-prime adaptor, is then ligated onto the library fragments. This adaptor contains a unique molecule ID, or UMI, which is a 12-basepair, randomized sequence that is unique to each molecule in the mixture.

8. Once both adaptors have ligated, the loop adaptor is cleaved.

9. The library is then PCR amplified to create the final, sequencer-ready library. During PCR, the sample index is added to the 3-prime adaptor through the PCR primer to enable pooling of samples prior to sequencing. PCR separates the library molecules from the beads. Note that only the original strand of DNA is carried through this PCR, and the bait strand is not present in the final library.

The resulting molecules provide highly-uniform coverage of target molecules with uniform 3-prime ends and variable 5-prime ends for each strand that is captured. The variable 5-prime ends are used in conjunction with the UMI to identify duplicate reads created during PCR amplification, improving the ability to assess the frequency of variants found in sequence reads.

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