Nanopore-based random genomic sampling for intraoperative diagnosis of brain tumors and beyond

Abstract Copy number variations (CNVs) are almost ubiquitous in cancer. In many cases, somatic CNV analysis has led to the identification of oncogenic pathways and molecular-defined therapeutic targets. Here, we develop iSCORED — a one-step random genomic DNA reconstruction method that enables efficient, unbiased quantification of CNVs using real-time nanopore sequencing. By leveraging the longconcatenated reads, we generate approximately 1–2 million genomic fragments within one hour of MinION sequencing, allowing for high-resolution genomic dosage comparisons. In our retrospective cohort of 26 banked malignant brain tumors, we demonstrated 100% concordance in CNV detections, including chromosomal alterations and oncogene amplifications when compared to clinically validated assays. The iSCORED sparse methylation profiling allows for concurrent brain tumor methylation classification and promoter methylation characterization in amplified oncogenes. We further implemented the pipeline to investigate a prospective cohort of 15 diagnostically challenging primary brain tumors with upgraded PromethION Flow Cells. The results showed 100% concordance in aberrant CNV detection, including diagnostic chromosomal gains/losses and oncogene amplifications. Additionally, 14/15 brain tumor methylation classifications aligned with final pathological diagnosis. The entire workflow, from tissue arrival to automatic generation of CNV and methylation reports, can be accomplished within 105 minutes. We have extended the application of our tool to various cancer types that require either CNV analysis for accurate diagnosis or actionable oncogene amplifications for proper therapeutics. Our ultrafast molecular analysis can enhance clinical decision-making, optimize surgical planning, and identify potential molecular therapies within surgical timeframes. Biography Chun-Chieh Lin is a neuropathologist and Assistant Professor at Dartmouth Health. His laboratory focuses on technology development and nanopore sequencing research to facilitate ultrafast molecular diagnosis of cancer.