HANDLE Technology
The HANDLE (Halo-Shape ANnealing and Defer-Ligation Enrichment) technology of the AmoyDx® HANDLE NGS kits enables a fast and flexible preparation of NGS libraries in only 5-6 hours with one hour hands-on time. This technology uses DNA probes that enable a PCR-free isolation of the genomic target regions to be sequenced via an extension-ligation reaction. All reactions take place in one reaction tube per sample, minimizing the risk of sample mix-ups.
Advantages of the HANDLE Technology
Fast and flexible protocol: Library preparation within one working day with multiple stop points
High safety: Only one reaction tube per sample minimizes the risk of sample mix-ups, even when analyzing DNA & RNA
Low workload: Only one PCR purification at the end of library preparation
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
Would you like to learn more?
A HANDLE probe consists of two DNA elements that are complementary to the flanking regions of the target region in the genome and are connected by a loop. For optimal sequencing of FFPE material, the regions covered by the probes are on average only about 120 nucleotides long. Each probe has a unique UID (Unique IDentifier) sequence, which is used to bioinformatically eliminate post-sequencing artifacts that arise during library amplification and sequencing.
The probes first hybridize with their complementary ends to the target regions of the genomic DNA. In some kits, gene fusions can also be detected at RNA level. For this purpose, the RNA is first transcribed into cDNA and then hybridized with the probes in a reaction tube together with the DNA. Probes for the detection of gene fusions at RNA level are not connected by a loop to allow a combination of all potential probe partners and thus a more comprehensive analysis of gene fusions. During the extension-ligation reaction, the probes are extended at their 3'-ends by a polymerase along the target DNA with complementary nucleotides, resulting in a copy of the target region. A ligase ligates the copied sequences with the 5' ends of the probes, thus generating circular DNA molecules. Remaining linear DNA strands (template DNA, cDNA and non-hybridized probes) are removed by a subsequent nuclease digestion. The remaining ring-shaped DNA molecules are amplified by a PCR reaction with a few cycles. At the same time, index sequences and binding sites for the sequencing primers and for the flow cell are added to the libraries via the PCR primers. Finally, the libraries are purified using magnetic beads in the single purification step of the HANDLE protocol.
All reactions from probe hybridization to PCR take place in one reaction tube per sample, thus minimizing the risk of sample mix-ups.
