(10) Comprehensive Genomic Investigation of Pediatric Cancers and Rare Genomic Disorders Using Oxford Nanopore Technologies (ont)

Background: Pediatric cancers and genetic disorders arise from diverse genomic and epigenomic alterations, including point mutations, copy number variations (CNVs), structural variations (SVs) such as translocations, aneuploidy (e.g., Down syndrome), single nucleotide variations (SNVs), insertions/deletions (indels), imprinting defects (e.g., Prader-Willi syndrome), and epigenetic changes like DNA methylation. While Illumina’s short-read next-generation sequencing (NGS) excels at detecting SNVs and indels, it struggles with SVs, CNVs, and native base modifications without additional methods like bisulfite conversion. Oxford Nanopore Technology (ONT) sequencing, with recent advances in accuracy and affordability, provides a comprehensive long-read platform for precision medicine in pediatric cancers and genetic disorders.

Objectives: This study aims to evaluate the utility of ONT as an alternative method to elucidate patterns associated with somatic and germline pathologies.

Design/Methods: We assessed ONT’s diagnostic capabilities in both areas. For germline disorders, ONT resolved a 14-year diagnostic odyssey for a patient with Gitelman-like syndrome and clarified diagnoses in cases with ambiguous Illumina whole-genome or whole-exome sequencing results. In pediatric chordomas, ONT revealed genome-wide patterns, including allele-specific methylation, CNVs, SNVs/indels, and mitochondrial DNA mutations.
ONT sequenced data were analyzed using bioinformatic tools like Dorado, Remora, Clair3, Spectre, Straglr, Modkit, Minimap2, and Samtools for basecalling, alignment, variant detection, and methylation profiling. Custom Perl and R pipelines supported downstream analyses and statistical correlations across samples.

Results: Our results underscore ONT’s ability to resolve haplotype-dependent patterns, SVs, CNVs, and methylation profiles, capturing genomic and epigenomic landscapes in a single assay

Conclusions: We are extending ONT analysis to additional pediatric tumors, including retinoblastoma, other solid tumors, and Formalin-Fixed Paraffin-Embedded (FFPE) samples, as well as genomic disorders like primary mitochondrial disorders (PMDs). With its high-coverage, cost-effective long-read data, ONT is poised to transform pediatric precision medicine.