ddCAP Technology
The ddCAP (dual-directional CAPture) method is based on the hybrid capture technology, in which the target regions of the original DNA are enriched using biotinylated probes. Since only a single sequence-specific probe is used for hybridization instead of two primers, as is usual with amplicon-based methods, the ddCAP method enables a very comprehensive analysis of gene fusions. In addition, compared to amplicon-based methods, a larger number of target regions can be sequenced in parallel. Combining the libraries before the capture reaction, as is the case with the ddCAP method, also enables very uniform sequencing of the libraries and therefore better resolution of variants. The method is therefore very well suited for sequencing cfDNA from liquid biopsies.
Advantages of the ddCAP Technology
Comprehensive analysis: Detection of unknown fusion partners
High sensitivity: Suitable for liquid biopsy samples
High accuracy: Use of UID sequences for efficient identification of PCR errors
Flexible use: All AmoyDx® NGS assays can be combined in one sequencing run and are suitable for many common Illumina sequencing platforms
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To prepare genomic DNA, the DNA is first fragmented using enzymes or ultrasound. In the next step, the end repair, overhanging ends of the DNA fragments are filled by a polymerase to create blunt ends.
The adapters with PCR primer binding sites are then ligated to the ends of the DNA fragments by a ligase. The adapters are also given unique UID (Unique IDentifier) sequences, which are used after sequencing to bioinformatically eliminate artifacts that arise during amplification of the library and sequencing. The DNA fragments are then amplified using a PCR with a few cycles, during which the index sequences and the binding sites for the sequencing primers and the flow cell are added.
After purification using magnetic beads, the samples are combined in a reaction tube. For the hybrid capture reaction, the DNA is hybridized overnight with biotinylated probes that are complementary to the target regions of the starting DNA. In the subsequent purification by Sepharose beads, the "capture" step, the fragments containing the target regions of the panel are enriched. After one more PCR and a subsequent purification, the libraries can be sequenced.
In the AmoyDx® FGFR1-4 NGS Panel, RNA for fusion detection is transcribed into cDNA at the beginning of the workflow and combined with the fragmented DNA before the end-repair step (DNA/cDNA co-library workflow).
