From Sanger to Oxford Nanopore MinION Technology: The Impact of Third-Generation Sequencing on Genetic Hematological Diagnosis

Abstract

Background: Sanger sequencing remains the gold standard for characterizing genetic variants in short DNA fragments (<700 bp). However, the increasing demand for short TATs and high sensitivities in variant detection, particularly in oncohematology, is driving the need for more efficient methods. Next-generation sequencing (NGS) has improved sensitivity and allows for the simultaneous analysis of multiple genes, but it is still costly and time-consuming. Consequently, Sanger sequencing continues to be widely used. In this study, we have compared Sanger sequencing with Oxford Nanopore technology (ONT), which offers enhanced sensitivity and faster sequencing, delivering diagnostic results within 24 h.

Methods: This study involves 164 samples (for a total of 174 analyzed regions of interest) previously characterized using either Sanger sequencing or a next-generation sequencing (NGS) panel, categorized by their genetic alterations. Validation was conducted on 15 genes crucial for the diagnosis, prognosis, or identification of drug resistance in myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). The primary objective was to assess whether MinION could identify the same variants previously detected in these patients.

Results and conclusions: With a 99.43% concordance observed in our comparison, our results support the implementation of MinION technology in routine variant detection in MPN, MDS, AML, and CML cases due to its significant advantages over Sanger sequencing.

Keywords: B-cell chronic lymphocytic leukemia; MinION technology; Oxford Nanopore MinION device; Sanger sequencing; acute myeloid leukemia; chronic myeloid leukemia; mutational analysis; myelodysplastic syndromes; myeloproliferative neoplasms; oncohematology.

Figure 1

(A) Workflow for the use of MinION as a diagnostic tool in oncohematology. Steps 5, 6, and 7 require the use of MinKNOW software and are necessary for sequencing analyses. (B) Schematic figure showing the library preparation process for sequencing with MinION. The use of barcodes is necessary in the case of multiplexing, where multiple samples will be sequenced at the same time in a single sequencing run. All the reagents required for these steps are provided by ONT in their sequencing kits. (Figure 1 has been created manually using the BioRender application (“https://www.biorender.com/”. Accesed date 23 May 2025).

Figure 2

Correlation plots between current gold standard techniques, Sanger (A) and NGS targeted panel (B) and our proposed method for clinical routine. Each point refering to a unique sample with VAF values using both technologies with a 5% of deviance shadow.