Targeted Genomic and Epigenomic Dual Analysis of the Same Region for an Entire Gene in Tumor DNA

Importance of Topic

Simultaneous genomic and epigenomic profiling of tumor DNA is crucial for comprehensive cancer characterization. Traditional approaches require separate assays or deep sequencing, limiting throughput and increasing costs. Targeted dual analysis of the same gene region addresses these challenges by preserving methylation marks and enabling mutation detection from minimal input.

Objectives and Study Overview

The study aimed to develop a robust workflow using the Agilent Avida Duo system to capture both DNA variants and methylation from the entire ARID1A gene within a single sample. Performance of the modified same-region capture was compared to the standard dual-region method, varying panel complexity and DNA fragmentation strategies.

Methodology and Instrumentation

The workflow began with 10 ng of gDNA, fragmented by either Covaris sonication or enzymatic shearing. Two custom 90 kb panels were designed: one targeting the Watson strand for variant detection and one targeting the Crick strand for methylation, mixing high-uniqueness (MT) and low-uniqueness (LT) probes at 4:4 and 4:2 ratios. Sequential capture used the flow-through from the first reaction for secondary enrichment. Sequencing was performed with dedicated read budgets: 10 M read pairs for DNA panels and 5 M for methylation panels. Control experiments utilized Agilent Avida Focused Cancer and custom methylation panels. Instrumentation included Agilent Avida Duo enrichment kits, Covaris S2, enzymatic shearing reagents, and Illumina sequencing platforms.

Main Results and Discussion

• The same-region capture approach achieved uniform coverage across high-GC and intronic regions, with the 4:2 MT/LT mix yielding slightly higher on-target rates and recovery than 4:4.
• Enzymatic shearing provided improved target recovery but marginally lower specificity than sonication.
• Methylation conversion efficiency remained consistently above 98.9% across all conditions.
• Analysis of four breast tumor samples revealed multiple ARID1A variants, including frameshift and stop-gain mutations, and distinct methylation patterns across 1366 CpG sites, with median CpG coverage between 97X and 570X.

Benefits and Practical Applications

This dual-capture workflow conserves limited samples by eliminating the need for splitting, reduces sequencing costs through optimized read allocation, and enhances data quality in challenging genomic regions. It is adaptable for low-input applications such as cfDNA and supports customizable target design, making it suitable for research and clinical laboratories focusing on cancer genomics and epigenetics.

Future Trends and Potential Applications

• Extension of the same-region dual analysis to additional cancer-related genes and noncoding regions.
• Integration with broader multiomic platforms for simultaneous RNA and protein analyses.
• Adaptation for liquid biopsy workflows to monitor tumor evolution and treatment response.
• Automation and scaling to high-throughput formats for large-cohort studies.

Conclusion

The Agilent Avida Duo methodology demonstrates a viable strategy for combined genomic and methylation profiling of the ARID1A gene from minimal DNA input. The approach offers flexibility, efficiency, and improved coverage uniformity, establishing a foundation for advanced multiomic cancer diagnostics.

Reference

• Tang W Zhang F Byun JS et al Population-specific Mutation Patterns in Breast Tumors from African American European American and Kenyan Patients Cancer Res Commun 2023 3 11 2244-2255