Background

The Homologous Recombination Repair (HRR) pathway plays an important role in double strand break, which is the major cause of cancer development. It has been demonstrated that loss of function of HRR genes (e.g. BRCA1, BRCA2, PALB2) and homologous recombination deficiency (HRD) will cause a higher risk of developing cancer, and patients with HRR gene mutations showed higher response to PARPi and platinum-containing therapies.

Intended Use

The AmoyDx^® HANDLE HRR NGS Panel (Reversible Terminator Sequencing) is intended for qualitative detection of single nucleotide variants (SNVs) and insertions and deletions (Indels) variants in protein coding regions and intron/exon boundaries of 27 HRR genes (AR, ATM, ATR, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CDK12, CHEK1, CHEK2, ESR1, FANCA, FANCL, HDAC2, HOXB13, MRE11, NBN, PALB2, PPP2R2A, PTEN, RAD51B, RAD51C, RAD51D, RAD54L, STK11 and TP53), and SNVs/Indels variants in hotspot regions of 5 driver genes (BRAF, ERBB2, KRAS, NRAS and PIK3CA) using DNA isolated from peripheral whole blood samples, fresh-frozen tumor tissue or formalin-fixed paraffin embedded (FFPE) tumor tissue specimens. In addition, the kit also allows the detection of large rearrangements (LRs) of the BRCA1 and BRCA2 genes from blood-derived DNA.
The kit is intended to be used by trained professionals in a laboratory environment.

Principles of the Procedure

The test kit is based on Halo-shape ANnealing and Defer-Ligation Enrichment system (HANDLE system) technology which is 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 DNA extracted from tissue or blood samples, and it offers a time saving protocol that can be completed within 5 hours, and requires just about 1 hour of hands-on time.

Figure 1. Principle of library construction (HANDLE system)

The probe contains an extension arm and a ligation arm which are complementary to the target gene region. Firstly, the probe anneals onto the DNA template of the target region. Secondly, the DNA is extended from the extension arm to the ligation arm with the help of DNA polymerase, then the nicks are repaired to generate the circular products with the help of DNA ligase. Next, the remaining linear probes, single-strand and double-strand DNA are digested with the help of enzyme exonuclease, and only the target circular DNA will be kept for PCR amplification. Finally, the universal PCR amplification is performed to enrich the target DNA, 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.

To order, please contact sales@amoydx.com.