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AmoyDx - HANDLE Classic NGS Panel
The AmoyDx HANDLE Classic NGS Panel (Reversible Terminator Sequencing) is a multi-biomarker next generation sequencing (NGS) assay that enables the detection of variants in 40 key solid tumor genes and microsatellite instability (MSI) status using DNA and RNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens. The assay allows concurrent analysis of DNA and RNA to simultaneously detect single nucleotide variants (SNVs), insertions and deletions (InDels), fusions, copy number amplifications (CNAs) and MSI in a single workflow. The kit is intended to be used by trained professionals in a laboratory environment.
The AmoyDx HANDLEThe test kit is based on Halo-shape ANnealing and Defer-Ligation Enrichment system (HANDLE system) technology which is an improved Molecular Inversion Probe (MIP) technology to capture the target gene region (Figure 1). During the library construction process, each individual DNA molecule is tagged with a unique molecular index (UMI) at both ends, which allows high sensitivity in variant detection by eliminating any library amplification and sequencing bias.
The test kit uses both DNA and RNA extracted from FFPE samples, and it offers a time saving protocol that can be completed within 6 hours, and requires just about 1 hour of hands-on time. Firstly, the RNA is reverse transcribed into cDNA with help of the Reverse Transcriptase and the RT primers. Secondly, the cDNA product and the genomic DNA are combined together in one tube for hybridization. The probe contains an extension arm and a ligation arm which are complementary to the target gene region, and the probe anneals onto the DNA or cDNA template of the target region. Thirdly, the DNA is extended from the extension arm to the ligation arm with help of the DNA polymerase, then the nicks are repaired to generate the circular products with help of the DNA ligase. Next, the remaining linear probes, single-strand and double-strand DNA are digested with help of the exonuclease, and only the target circular DNA will be kept for PCR amplification. Finally, the universal PCR amplification is performed to enrich the target libraries, and the magnetic bead-based purification is performed to obtain the final library.
After quality control (QC), the qualified libraries could be sequenced on Illumina sequencing platform. The sequencing data can be analyzed by AmoyDx NGS data analysis system (ANDAS) to detect the genomic variants in the target region.
- High Ability - Detect SNV, InDel, Fusion, CNA and MSI simultaneously.
- Limit of Detection (LoD) - SNV/Indel (1% MAF), Fusion (100 copies) , CNV (5 copy number).
- Less samples - DNA & RNA co-detection in one workflow.
- High Efficiency - 6 hours TAT for library preparation and 1 hour for hands-on time only.