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...
| Authors: | ; ; ; ; ; ; |
|---|---|
| Format: | Article |
| Publication date: | 2025 |
| Country: | España |
| Institution: | Conselleria de Salut i Consum del Govern de les Illes Balears |
| Repository: | Docusalut |
| Language: | English |
| OAI Identifier: | oai:docusalut.com:20.500.13003/25537 |
| Online Access: | https://hdl.handle.net/20.500.13003/25537 |
| Access Level: | Open access |
| Keyword: | Leukemia, Myeloid, Acute Leucemia Mieloide Aguda AML FLT3-ITD TGS acute myeloid leukemia minION third-generation sequencing |
| _version_ | 1878731383706222592 |
|---|---|
| author | Martínez-Serra, Jordi |
| author2 | Alonso-Carballo, Aser Horrillo, Ángel Gomez Fernandez, Paula Vögler, Oliver Gutiérrez, Antonio Sampol Mayol, Antonia |
| author2_role | author author author author author author |
| author_browse | Alonso-Carballo, Aser Gomez Fernandez, Paula Gutiérrez, Antonio Horrillo, Ángel Martínez-Serra, Jordi Sampol Mayol, Antonia Vögler, Oliver |
| author_facet | Martínez-Serra, Jordi Alonso-Carballo, Aser Horrillo, Ángel Gomez Fernandez, Paula Vögler, Oliver Gutiérrez, Antonio Sampol Mayol, Antonia |
| author_role | author |
| collection | Docusalut |
| 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. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | ES_2e76ca72ccba2ca6249fbbeef10a00f7 |
| institution | Conselleria de Salut i Consum del Govern de les Illes Balears |
| instname_str | Conselleria de Salut i Consum del Govern de les Illes Balears |
| language | eng |
| network_acronym_str | ES |
| network_name_str | España |
| oai_identifier_str | oai:docusalut.com:20.500.13003/25537 |
| publishDate | 2025 |
| publishDateSort | 2025 |
| publisher | Taylor & Francis |
| publisherStr | Taylor & Francis |
| reponame_str | Docusalut |
| spelling | 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 |
| 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 | 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_short | 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 |
| 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 |
| url | https://hdl.handle.net/20.500.13003/25537 |