Technology Spotlight
Long-Read Sequencing for Hematologic Malignancies Free

Genomic characterization of somatic variants is central to the diagnosis, classification, and risk stratification of hematologic malignancies. Traditionally, this has relied on a fragmented pipeline of cytogenetics, fluorescence in situ hybridization, targeted polymerase chain reaction (PCR), and next-generation sequencing. While powerful, these methods are often slow, expensive, and incomplete. Long-read sequencing (LRS), often called third-generation sequencing, offers the potential to unify and accelerate genomic diagnostics across hematology.

Unlike short-read sequencing, which produces DNA fragments of less than 600 base pairs, LRS generates reads tens of thousands of bases long. This allows for direct resolution of genomic structural variants, copy number alterations, and repetitive elements. Two major platforms currently dominate the field:

• PacBio — uses single-molecule, real-time sequencing with high-fidelity reads, achieving high per-base accuracy and robust methylation calling

• Oxford Nanopore Technologies (ONT) — uses nanopore membranes and ionic current disruptions to sequence nucleotides in real time, enabling portability, adaptive sampling (computational target enrichment), and direct detection of base modifications

Critically, these platforms can be used without PCR amplification, preserving native DNA and RNA modifications. As a result, LRS enables interrogation of genomes, transcriptomes, and epigenomes, often without using multiple assays.

Initial adoption of LRS focused on resolving rare Mendelian disorders and structural variant detection. More recently, applications in oncology — including hematologic malignancies — have demonstrated its clinical potential:

• Acute myeloid leukemia (AML) — A nanopore-based assay targeting common AML genes (e.g., NPM1, FLT3, CEBPA, IDH1/2, and TP53) reduced turnaround time for mutation profiling from one week to under 24 hours at costs as low as $200.^1,2 

• Fusion detection — Nanopore sequencing can rapidly identify oncogenic fusions such as BCR-ABL1, which are critical for risk stratification and therapy choice. One study achieved fusion transcript detection in acute leukemia within 15 minutes of sequencing.^2,3 

• Epigenomics — Nanopore sequencing enables direct methylation profiling without bisulfite conversion, distinguishing tumor entities such as brain tumors and offering similar promise in leukemia.⁴ 

Adaptive sampling leverages nanopore’s real-time read rejection capability to enrich for target regions without additional wet lab steps. This strategy increases depth over leukemia-associated loci manyfold while maintaining genome-wide breadth. The result is comprehensive classification — from digital karyotyping to single-nucleotide variant detection — within a single assay.

In a prospective study of 57 pediatric acute leukemia cases, ONT whole-genome sequencing with adaptive sampling identified subtype-defining alterations — aneuploidy, translocations, copy number variations, and cryptic fusions — with 96% sensitivity and 100% specificity compared to standard clinical methods. Crucially, clinically actionable fusions such as ETV6::RUNX1, KMT2A rearrangements, and DUX4 rearrangements were resolved within hours, and sometimes in as little as 15 minutes.⁵ 

Similarly, at the University of Tokyo, investigators applied adaptive sampling to 28 children with AML, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL, targeting 466 leukemia-associated genes. Subtype-defining alterations were identified in 86% of cases, including cryptic rearrangements such as DUX4 fusions that escaped standard clinical testing. Adaptive sampling also efficiently captured chromosome-level copy number variations and germline predisposition variants while reducing turnaround to approximately 72 hours.⁶ 

LRS is poised to reshape the diagnostic landscape for hematologic malignancies:

• Single-assay diagnostics — Replacement of current fragmented workflows with a single comprehensive test

• Rapid turnaround — Potential for same-day genomic diagnosis, guiding induction therapy in acute leukemia where timing is critical

• Integrated multiomics — Concurrent genome, methylome, and chromatin state within the same assay

• Global accessibility — Portable sequencers (e.g., ONT MinION) with the potential to democratize leukemia diagnostics in resource-limited settings

• Residual disease and monitoring — Direct fusion breakpoint tracking and methylation-based signatures with the potential to enhance minimal residual disease detection beyond flow cytometry or PCR

LRS is transitioning from research innovation to clinical reality in hematology. Its ability to rapidly and comprehensively characterize the diverse genomic alterations of leukemia offers a path toward more precise, efficient, and equitable cancer care. As accuracy improves and costs decline, LRS is likely to become a mainstay of hematologic malignancy diagnostics worldwide.

Dr. Furlan indicated no relevant conflicts of interest.