Third‐Generation Nanopore Sequencing for Post‐Transplant Chimerism Monitoring

ABSTRACT Post‐transplant chimerism monitoring is a cornerstone of hematopoietic stem cell transplantation (HSCT) follow‐up, providing early and clinically actionable information on engraftment, graft rejection and disease relapse. Analytical strategies have progressively evolved from STR‐based assays towards more sensitive molecular approaches, including qPCR, dPCR and next‐generation sequencing (NGS). Although NGS offers high analytical robustness and multiplexing capacity, its routine use remains limited by turnaround time and batch‐dependent workflows, restricting its applicability in urgent clinical contexts. Third‐generation sequencing (TGS), based on real‐time DNA sequencing, emerges as a complementary approach combining molecular sensitivity with enhanced operational flexibility. We evaluated ONtrack (Eurobio/GenDx), the first commercial TGS‐based assay for post‐transplant chimerism quantification using Oxford Nanopore Technologies, currently available through an early access program. Thirty samples were analysed, including external quality assessment materials, manufacturer controls and clinical specimens from three transplanted patients. Sample types included whole blood, bone marrow and CD3 + ‐sorted cell fractions. ONtrack results were compared with routinely used methods (qPCR, dPCR and NGS). ONtrack demonstrated excellent analytical performance, with strong correlations to qPCR ( R 2 = 0.955), dPCR ( R 2 = 0.990) and NGS ( R 2 = 0.998). Mixed chimerism was reliably detected down to 0.5%, and Bland–Altman analyses confirmed good agreement across methods. Z‐score analysis showed satisfactory performance for all evaluated samples. Notably, the complete analytical workflow could be completed in approximately 3 h for a single sample, with fully automated data processing and integrated quality controls. Beyond analytical accuracy, ONtrack offers high operational versatility, enabling both urgent single‐sample testing and batch analyses using a standardised workflow. Although the limited number of markers may restrict detection of ultra‐low microchimerism, TGS represents a valuable complementary solution for early post‐transplant monitoring, particularly in decentralised laboratory settings.