Optimization of a hybridization-based target enrichment protocol for precision oncology

Area of Science:

• Genomics
• Molecular Biology
• Oncology

Background:

• Next-generation sequencing (NGS) is crucial for precision oncology, detecting genomic alterations.
• Formalin-fixed paraffin-embedded (FFPE) samples are challenging for DNA analysis.
• Optimizing enrichment protocols is vital for FFPE-derived DNA.

Purpose of the Study:

• To optimize a hybridization-based enrichment protocol for FFPE DNA.
• To evaluate its performance against commercial kits.
• To ensure comprehensive genomic alteration detection in FFPE samples.

Main Methods:

• Systematic variation of hybridization conditions (temperature, duration, buffer, capture rounds).
• Sequencing libraries using Illumina platforms.
• Comparative analysis against commercial hybridization- and amplification-based kits and whole-exome capture panels.

Main Results:

• Two rounds of short hybridization maximized enrichment efficiency and coverage uniformity.
• The optimized protocol showed sixfold higher enrichment than the analogue.
• Scalability testing demonstrated performance comparable to a commercial whole-exome kit.

Conclusions:

• The optimized protocol offers superior target coverage and high variant recall for FFPE samples.
• It provides flexibility for scaling from small panels to whole-exome sequencing.
• The protocol is suitable for precision oncology applications using challenging FFPE samples.