Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection
BACKGROUND: Internal tandem duplications (FLT3-ITDs) in the FLT3 gene constitute a key driver mutation in acute myeloid leukemia (AML), strongly associated with poor prognosis and therapeutic resistance. Although general-purpose structural variant callers such as Sniffles have been used to detect FL...
| Autores: | , , , , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 2025 |
| País: | España |
| Institución: | Conselleria de Salut i Consum del Govern de les Illes Balears |
| Repositorio: | Docusalut |
| Idioma: | inglés |
| OAI Identifier: | oai:docusalut.com:20.500.13003/25537 |
| Acceso en línea: | https://hdl.handle.net/20.500.13003/25537 |
| Access Level: | acceso abierto |
| Palabra clave: | Leukemia, Myeloid, Acute Leucemia Mieloide Aguda AML FLT3-ITD TGS acute myeloid leukemia minION third-generation sequencing |
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Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection Martínez-Serra, Jordi Alonso-Carballo, Aser Horrillo, Ángel Gomez Fernandez, Paula Vögler, Oliver Gutiérrez, Antonio Sampol Mayol, Antonia Leukemia, Myeloid, Acute Leucemia Mieloide Aguda AML FLT3-ITD TGS acute myeloid leukemia minION third-generation sequencing BACKGROUND: Internal tandem duplications (FLT3-ITDs) in the FLT3 gene constitute a key driver mutation in acute myeloid leukemia (AML), strongly associated with poor prognosis and therapeutic resistance. Although general-purpose structural variant callers such as Sniffles have been used to detect FLT3-ITDs, their limitations in resolving clonal diversity and low-frequency variants can lead to underrepresentation of minor clones. These shortcomings highlight the need for a dedicated bioinformatics pipeline. MATERIALS AND METHODS: We developed a custom clustering-based pipeline to overcome the constraints of generic SV callers, leveraging Oxford Nanopore's MinION for sequencing. Our method focuses on FLT3-ITDs by grouping near-identical insertions into biologically meaningful subclones, thereby allowing accurate variant detection of even low-frequency events. The pipeline was benchmarked against capillary electrophoresis (CE) and Sniffles at various thresholds (including 10%, 20%, and 50% allele-frequency cutoffs), with results validated via IGV inspection and cross-mapping. RESULTS: The pipeline successfully detected FLT3-ITDs across all tested samples, including low-frequency variants and diverse subclones that Sniffles overlooked. Analyses uncovered complex multiclonal architectures composed of dominant clones (~20-25% of reads) plus multiple minor subclones differing in length, sequence, and breakpoint. Crucially, our approach identified duplications as short as 15 bp-events often dismissed by conventional SV callers. Comparative analyses showed that Sniffles failed to call several biologically validated ITDs detected by our custom pipeline. CONCLUSION: Third-generation sequencing combined with a tailored clustering strategy enhances the detection of FLT3-ITDs and clonal diversity in AML compared to generic variant callers. This method provides critical insights into subclonal populations driving relapse and therapeutic resistance-particularly in relapsed/refractory AML-underscoring the importance of specialized pipelines for precision medicine in leukemia. Taylor & Francis https://hdl.handle.net/20.500.13003/25537 |
| title |
Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection |
| spellingShingle |
Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection Martínez-Serra, Jordi Leukemia, Myeloid, Acute Leucemia Mieloide Aguda AML FLT3-ITD TGS acute myeloid leukemia minION third-generation sequencing |
| title_short |
Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection |
| title_full |
Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection |
| title_fullStr |
Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection |
| title_full_unstemmed |
Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection |
| title_sort |
Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection |
| author |
Martínez-Serra, Jordi |
| author_facet |
Martínez-Serra, Jordi Alonso-Carballo, Aser Horrillo, Ángel Gomez Fernandez, Paula Vögler, Oliver Gutiérrez, Antonio Sampol Mayol, Antonia |
| author_role |
author |
| author2 |
Alonso-Carballo, Aser Horrillo, Ángel Gomez Fernandez, Paula Vögler, Oliver Gutiérrez, Antonio Sampol Mayol, Antonia |
| author2_role |
author author author author author author |
| topic |
Leukemia, Myeloid, Acute Leucemia Mieloide Aguda AML FLT3-ITD TGS acute myeloid leukemia minION third-generation sequencing |
| topic_facet |
Leukemia, Myeloid, Acute Leucemia Mieloide Aguda AML FLT3-ITD TGS acute myeloid leukemia minION third-generation sequencing |
| description |
BACKGROUND: Internal tandem duplications (FLT3-ITDs) in the FLT3 gene constitute a key driver mutation in acute myeloid leukemia (AML), strongly associated with poor prognosis and therapeutic resistance. Although general-purpose structural variant callers such as Sniffles have been used to detect FLT3-ITDs, their limitations in resolving clonal diversity and low-frequency variants can lead to underrepresentation of minor clones. These shortcomings highlight the need for a dedicated bioinformatics pipeline. MATERIALS AND METHODS: We developed a custom clustering-based pipeline to overcome the constraints of generic SV callers, leveraging Oxford Nanopore's MinION for sequencing. Our method focuses on FLT3-ITDs by grouping near-identical insertions into biologically meaningful subclones, thereby allowing accurate variant detection of even low-frequency events. The pipeline was benchmarked against capillary electrophoresis (CE) and Sniffles at various thresholds (including 10%, 20%, and 50% allele-frequency cutoffs), with results validated via IGV inspection and cross-mapping. RESULTS: The pipeline successfully detected FLT3-ITDs across all tested samples, including low-frequency variants and diverse subclones that Sniffles overlooked. Analyses uncovered complex multiclonal architectures composed of dominant clones (~20-25% of reads) plus multiple minor subclones differing in length, sequence, and breakpoint. Crucially, our approach identified duplications as short as 15 bp-events often dismissed by conventional SV callers. Comparative analyses showed that Sniffles failed to call several biologically validated ITDs detected by our custom pipeline. CONCLUSION: Third-generation sequencing combined with a tailored clustering strategy enhances the detection of FLT3-ITDs and clonal diversity in AML compared to generic variant callers. This method provides critical insights into subclonal populations driving relapse and therapeutic resistance-particularly in relapsed/refractory AML-underscoring the importance of specialized pipelines for precision medicine in leukemia. |
| publishDate |
2025 |
| format |
article |
| url |
https://hdl.handle.net/20.500.13003/25537 |
| language |
eng |
| eu_rights_str_mv |
openAccess |
| publisher |
Taylor & Francis |
| institution |
Conselleria de Salut i Consum del Govern de les Illes Balears |
| collection |
Docusalut |
| reponame_str |
Docusalut |
| instname_str |
Conselleria de Salut i Consum del Govern de les Illes Balears |
| _version_ |
1878432969699360768 |
| publishDateSort |
2025 |
| author_browse |
Alonso-Carballo, Aser Gomez Fernandez, Paula Gutiérrez, Antonio Horrillo, Ángel Martínez-Serra, Jordi Sampol Mayol, Antonia Vögler, Oliver |
| publisherStr |
Taylor & Francis |
| score |
6,9303427 |