New! | Dark Probe technology improves HRD research – a new tool for precision medicine

Genomic stability can be maintained through Homologous Recombination Repair (HRR) when DNA double-strand breaks occur in normal cells, but mutations in HRR-related genes can cause Homologous Recombination Deficiency (HRD), which manifests as Genomic Scar (GS).

 

The detection of HRD can be used to assess the risk of cancers, including breast, ovarian, and pancreatic cancers, etc. Also, since most HRD tumors are highly sensitive to PARP inhibitors (PARPi) and platinum drugs, the results of HRD detection can be used to predict the efficacy and provide the clinical medication guidance.

 

Traditional strategy of differential capture

A comprehensive HRD assessment consists of variant detection of HRR-related genes and genomic scar identification based on SNPs loci. From the perspective of clinical testing accuracy, HRR gene detection and genomic scar identification based on SNPs loci generally require no less than 500x and 100x effective sequencing depths respectively, to detect mutations with a frequency of no less than 1% and SNPs loci with high MAF values in healthy Chinese populations.

 

 

For differential capture, the traditional strategy is to control the ratio of different probes during hybridization, but the molar concentration of probes relative to the template molecules is excessive, making it time-consuming and laborious to adjust the probe concentration ratio to achieve differential capture, and the results are unstable and do not achieve good isobaric scaling.

 

The Dark Probe technology makes differential capture visible and easy

Compared to traditional strategy, Dynegene's self-developed Dark Probe technology makes complex differential capture easier. The Dark Probe technology not only shortens the optimization period and saves cost and labor, but also optimizes the instability of results caused by the traditional strategy of controlling probe ratio, and has high intra-batch and inter-batch consistency.

 

 

More importantly, the high precision result can be easily scaled according to the customers’ various needs of differential capture, which is a promising application.

Workflow of Dark Probe

 

Product Highlights

Comprehensive testing

20 HRR-related core genes are selected based on NCCN guidelines and clinical trial experience; 50,000 SNPs with high MAF values are also collected to cover as many heterozygous loci as possible for genomic scar identification.

Shorter optimization period for custom needs

For differential capture needs, the Dark Probe technology significantly shortens the optimization period, and can easily achieve isobaric scaling according to various differential capture needs of customers.

Excellent Consistency

The Dark Probe technology optimizes the instability of results caused by the traditional strategy of controlling probe ratios, and provides excellent consistency for intra-batch and inter-batch.

Lower Cost

 

Differential capture directly leads to a significant reduction in sequencing costs.

 

Data demonstration

Good performance of capture

Input 100 ng of standard (NA12878) for ultrasonic shearing and DNA library construction (QuarPrep DNA Library Kit, Dynegene, NL1001); 750 ng of constructed library treated by conventional hybridization capture using QuarXeq HRD probe (QuarHyb One Reagent Kit, Dynegene, NC1003), three replicate experiments, sequencing volumn of 6G on illumina platform.

Figure 1. Capture Performance of QuarXeq HRD Probes

The ratio of HRR and HRD depth under differential capture is consistent with the expectation

 

Input 100 ng of standard (NA12878) for ultrasonic shearing and DNA library construction (QuarPrep DNA Library Kit, Dynegene, NL1001); 750 ng of constructed library treated by conventional hybridization capture using QuarXeq HRD Probes probe (QuarHyb One Reagent Kit, Dynegene, NC1003), three replicate experiments, illumina platform sequencing. The analysis results showed that the ratio of HRR depth to HRD depth was 5:1, as expected.

Figure 2. Depth under Differential Capture Using QuarXeq HRD Probes

 

The HRD BRCA2 depth and Chr 13 depth were also analyzed for the BRCA2 gene, and the results showed a ratio of 5:1, also as expected.

Figure 3. Comparison of HRD BRCA2 depth and CHR 13 depth under differential capture using QuarXeq HRD Probes

Uniform coverage of SNPs loci

 

The SNPs loci in QuarXeq HRD Probes are evenly distributed to cover as many heterozygous loci as possible, ensuring that LOH, LST, TAI that meet the requirements can be detected.

Figure 4. Coverage of SNP Marker chromosomes

Note: the gray area is the highly repetitive region on the reference genome, and the blue scatter is the SNP Marker MAF repeat element.

 

HRR-related genes

 

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