Technology

Step 1: Denature DNA

NexGenAbler Step 1: Denature cfDNA
cfDNA in 10 µL H2O.

Both single-stranded or double stranded DNA can be analyzed. If double-stranded, each strand is processed. The green dot represents the site of interest (e.g. mutation, indel, SNP).

Step 2: Dynamic Molecular Indexing and 3′ End Conditioning

NexGenAbler Step 2: Dynamic Molecular Indexing
Reaction volume is now 20 µL

Aliquots of Tube A and Tube B are mixed together and an equal volume is added to the reaction tube. A truly unique molecular index is added to each DNA strand and the 3’ ends are conditioned for high-efficiency ligation.

Step 3: Circularization

NexGenAbler Step 3: Circularization of cfDNA.
Reaction volume is now 40 µL

Aliquots of Tube C and Tube D are mixed together and an equal volume is added to the reaction tube. High-efficiency ligation results in circularization. Contaminating HMW lymphocyte DNA is automatically excluded.

Step 4: Exonuclease Treatment

NexGenAbler Step 4: cfDNA cleanup with exonuclease.
Reaction volume is now 46 µL

Six (6) µL of Tube E is added. Contaminating HMW lymphocyte DNA is digested away. Closed circular DNA (molecular-indexed cfDNA) is preserved.
Step 5: Inverse PCR

NexGenAbler Step 5: cfDNA amplification via Inverse PCR.
Reaction volume is now 92 µL

Aliquots of Tubes F, G, and Tube H are mixed together and an equal volume is added to the reaction tube. Inverse PCR copies over the molecular index and the site of interest. Samples are ready for clean up and optional sample indexing, then sequencing.

That’s it – five easy steps! 

The NexGenAnalyzer assembles sequencing results into read families based on their Dynamic Molecular Indexes (DMI), gene-specific 5’ end sequences, gene-specific 3’ end sequences, and total length (gene-specific length plus DMI length. Wild type or mutant calls are then made (or SNP calls, depending on your application). Calls and quantitation are made with precision.

Patents covering the NexGenAbler™ method have been allowed in the United States and Europe.