A Differential Depth Sequencing Method, SPRE-Seq, for Enhancing Targeted Region Coverage in Hybridization...

Area of Science:

• Genomics and Bioinformatics
• Molecular Diagnostics

Background:

• Sequencing depth is critical for accurate variant detection in next-generation sequencing (NGS).
• Capture-based targeted NGS faces limitations balancing sequencing breadth and depth.
• Homologous recombination deficiency (HRD) analysis requires precise sequencing of specific genomic regions.

Purpose of the Study:

• To introduce SPRE-Seq, a differential depth sequencing method for targeted NGS panels.
• To evaluate SPRE-Seq's performance in homologous recombination repair (HRR) and HRD region analysis.
• To assess SPRE-Seq's efficiency in reducing sequencing data volume without compromising accuracy.

Main Methods:

• Developed and implemented SPRE-Seq, a method enabling variable sequencing depths across targeted regions.
• Validated SPRE-Seq using a custom-designed HRD assay with HRD reference standards and clinical samples.
• Compared SPRE-Seq performance against regular capture methods in terms of data volume and accuracy.

Main Results:

• SPRE-Seq achieved required sequencing depths for HRR and HRD regions in reference standards using 50% less data (6 GB vs. 12 GB).
• 100% concordance was observed between SPRE-Seq results and expected outcomes for HRR genes and HRD status.
• Clinical sample analysis showed significantly higher effective depth in HRR regions with SPRE-Seq at 6 GB compared to regular capture at 12 GB, with no significant difference at 6 GB.

Conclusions:

• SPRE-Seq is a feasible and reliable method for determining HRD status and HRR somatic variants.
• The approach significantly reduces sequencing data volume while ensuring adequate sequencing depth.
• SPRE-Seq offers a cost-effective solution for targeted NGS applications requiring precise depth control.