Step 1: Denature DNA
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
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
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
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
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.