PACLseq: A Standalone Diagnostic Method for Ph-Like Acute Lymphoblastic Leukemia Using Nanopore Sequencing

Timely and accurate detection of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL)-related fusion gene is essential for treatment decisions. However, due to the complexity of possible gene fusion combinations of Ph-like ALL, current diagnostic workflows face critical limitations: prolonged turnaround (7-14 days), high costs, and deficiency in degraded specimens. In this study, we introduce Partial Anchored Capture and Long-Read Sequencing (PACLseq), a nanopore-sequencing-technology-based approach. We designed a detection panel associated with Ph-like ALL, specifically <i>ABL2</i>, <i>CSF1R</i>, <i>PDGFRB</i>, <i>JAK2</i>, <i>ABL1</i>, <i>EPOR</i>, and <i>CRLF2</i> as target genes. Validated on 47 clinical samples, PACLseq achieved 93.3% sensitivity and 100% specificity in 26 degraded RNA samples (RIN > 3). Crucially, PACLseq maintained detection accuracy in nine low-RIN samples (RIN ≤ 3) with fragmented transcripts. The method requires only 10 ng of RNA input, delivers results in 3 days (vs. 7-14 days for conventional methods), and reduces costs by 50%. By offering rapid and accurate fusion detection, PACLseq has the potential to significantly improve diagnostic efficiency, facilitate timely treatment decisions, and enhance patient outcomes in the management of Ph-like ALL.