73. Discovery and validation of genomic alterations by optical genome mapping and nanopore long-read sequencing

Human cancer cell lines serve as indispensable model systems for exploration of genomic alterations arising from genetic instability during oncogenesis as well as identification and validation of novel targets for diagnostics or therapeutics. The CCRF-CEM cell line, derived from a female patient with T-cell acute lymphoblastic leukemia (T-ALL), presents a complex genomic landscape. This study aims to leverage high-resolution optical genome mapping (OGM) and long-read sequencing to comprehensively characterize structural variants (SVs), copy number variations (CNVs), and single nucleotide variants (SNVs), while validating previously identified aberrations. A combination of cytogenetic and genomic approaches was applied, including karyotyping, fluorescent in situ hybridization (FISH), array comprehensive genomic hybridization (aCGH) using Oxford Gene Technology (OGT) and Affymetrix platforms, and OGM with the Saphyr system. For whole-genome sequencing (WGS), genomic DNA was extracted and libraries were prepared using either ultra-long (UL) DNA sequencing or rapid barcoding (RB) sequencing workflow from Oxford Nanopore Technologies (ONT). The PromethION 2 Solo/GridION sequencer was employed and sequencing reads > 1 kb were retained. MinKNOW was used to perform high accuracy basecalling (quality score ≥9) and passed sequencing reads (FASTQ files) were aligned to the GRCh38 human reference genome assembly using wf-alignment in EPI2ME. The resulting BAM files were visualized using the Integrative Genomics Viewer (IGV). Sequencing reads from nine independent runs were merged and grouped into two datasets – Group 1 (UL workflow) and Group 2 (RB and UL workflows). A total of 11.8 million reads were generated, aligning 100% to GRCh38 with cumulative sequencing yield exceeding 355 billion bases. Mean basecall accuracy reached a Q17.3 (Group 1) and Q16.4 (Group 2), whereas the mean read lengths were 39.9k (Group 1) and 22.4k (Group 2). Collectively, the mean value of alignment accuracy was ≥96.5% and coverage ≥96.4%. Comparative analysis confirmed a likely pathogenic TP53 missense mutation (c.743G>T, p.Q248L), previously detected by aCGH/Affymetrix. Notably, long-read sequencing validated key SVs detected via OGM, including an 81.9-kb deletion in STIL (1p33), a 226-kb deletion containing PTEN (10q23.31), and an unbalanced translocation der(5)t(5;14)(q35.2;q32.2). A second translocation, t(8;9)(p11;p24), identified via OGM, was not confirmed, warranting further breakpoint analysis. Additionally, a gain in chromosome 20 was detected, confirming OGM, aCGH and prior cytogenetic results. The integration of ONT long-read sequencing with OGM enables high-resolution mapping of structural and sequence-level genomic alterations in CCRF-CEM . The high accuracy of ONT’s basecalling and read alignment provides robust validation of key mutations, deletions, and translocations. Future work will focus on refining variant calling parameters, improving breakpoint resolution, and conducting functional assays to elucidate the biological impact of these aberrations in T-ALL pathogenesis. This study underscores the power of combining OGM and long-read sequencing for comprehensive genome characterization and clinical research applications.