Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Accurate detection of cancer-related gene mutations is crucial for targeted therapy.
• Long-read sequencing offers potential for comprehensive genomic analysis.
• Characterizing complex mutational patterns, like co-occurring mutations, remains a challenge.

Purpose of the Study:

• To evaluate the utility of long-read cDNA amplicon sequencing for characterizing diverse mutations in cancer-related genes.
• To assess the accuracy of long-read sequencing for single nucleotide variants (SNVs) and gene fusions.
• To investigate the application of long-read sequencing in phasing mutations and analyzing allelic backgrounds for therapeutic insights.

Main Methods:

• Employed MinION long-read portable sequencer for cDNA amplicon sequencing.
• Targeted sequencing of cancer-related genes: EGFR, KRAS, NRAS, and NF1.
• Performed serial dilution analysis to assess detection limits in low cancer cell populations.

Main Results:

• Achieved high precision (87.5%) and recall (91.3%) for homozygous SNVs.
• Reached 100% precision and recall for previously reported hotspot mutations.
• Successfully detected precise junctions of multiple gene fusions (e.g., EML4-ALK, CCDC6-RET).
• Phased EGFR mutations, revealing allelic backgrounds of drug-sensitive and resistant variants.
• Detected mutations in clinical samples, demonstrating real-world applicability.

Conclusions:

• Long-read sequencing provides a convenient and accurate method for comprehensive cancer mutation profiling.
• This approach can elucidate complex mutational landscapes, including phased mutations and allelic ratios.
• The technology holds promise for improving diagnostic and therapeutic strategies in oncology.