Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing.
Recurrent gene fusions are common drivers of disease pathophysiology in leukemias. Identifying these structural variants helps stratify disease by risk and assists with therapy choice. Precise molecular diagnosis in low-and-middle-income countries (LMIC) is challenging given the complexity of assays...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2023-01-01
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| Series: | PLOS Global Public Health |
| Online Access: | https://doi.org/10.1371/journal.pgph.0002267 |
| _version_ | 1797682736098443264 |
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| author | Olga Sala-Torra Shishir Reddy Ling-Hong Hung Lan Beppu David Wu Jerald Radich Ka Yee Yeung Cecilia C S Yeung |
| author_facet | Olga Sala-Torra Shishir Reddy Ling-Hong Hung Lan Beppu David Wu Jerald Radich Ka Yee Yeung Cecilia C S Yeung |
| author_sort | Olga Sala-Torra |
| collection | DOAJ |
| description | Recurrent gene fusions are common drivers of disease pathophysiology in leukemias. Identifying these structural variants helps stratify disease by risk and assists with therapy choice. Precise molecular diagnosis in low-and-middle-income countries (LMIC) is challenging given the complexity of assays, trained technical support, and the availability of reliable electricity. Current fusion detection methods require a long turnaround time (7-10 days) or advance knowledge of the genes involved in the fusions. Recent technology developments have made sequencing possible without a sophisticated molecular laboratory, potentially making molecular diagnosis accessible to remote areas and low-income settings. We describe a long-read sequencing DNA assay designed with CRISPR guides to select and enrich for recurrent leukemia fusion genes, that does not need a priori knowledge of the abnormality present. By applying rapid sequencing technology based on nanopores, we sequenced long pieces of genomic DNA and successfully detected fusion genes in cell lines and primary specimens (e.g., BCR::ABL1, PML::RARA, CBFB::MYH11, KMT2A::AFF1) using cloud-based bioinformatics workflows with novel custom fusion finder software. We detected fusion genes in 100% of cell lines with the expected breakpoints and confirmed the presence or absence of a recurrent fusion gene in 12 of 14 patient cases. With our optimized assay and cloud-based bioinformatics workflow, these assays and analyses could be performed in under 8 hours. The platform's portability, potential for adaptation to lower-cost devices, and integrated cloud analysis make this assay a candidate to be placed in settings like LMIC to bridge the need of bedside rapid molecular diagnostics. |
| first_indexed | 2024-03-12T00:04:09Z |
| format | Article |
| id | doaj.art-6e77bb1d6af84557843a38610a4ff84e |
| institution | Directory Open Access Journal |
| issn | 2767-3375 |
| language | English |
| last_indexed | 2024-03-12T00:04:09Z |
| publishDate | 2023-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLOS Global Public Health |
| spelling | doaj.art-6e77bb1d6af84557843a38610a4ff84e2023-09-17T06:18:02ZengPublic Library of Science (PLoS)PLOS Global Public Health2767-33752023-01-0139e000226710.1371/journal.pgph.0002267Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing.Olga Sala-TorraShishir ReddyLing-Hong HungLan BeppuDavid WuJerald RadichKa Yee YeungCecilia C S YeungRecurrent gene fusions are common drivers of disease pathophysiology in leukemias. Identifying these structural variants helps stratify disease by risk and assists with therapy choice. Precise molecular diagnosis in low-and-middle-income countries (LMIC) is challenging given the complexity of assays, trained technical support, and the availability of reliable electricity. Current fusion detection methods require a long turnaround time (7-10 days) or advance knowledge of the genes involved in the fusions. Recent technology developments have made sequencing possible without a sophisticated molecular laboratory, potentially making molecular diagnosis accessible to remote areas and low-income settings. We describe a long-read sequencing DNA assay designed with CRISPR guides to select and enrich for recurrent leukemia fusion genes, that does not need a priori knowledge of the abnormality present. By applying rapid sequencing technology based on nanopores, we sequenced long pieces of genomic DNA and successfully detected fusion genes in cell lines and primary specimens (e.g., BCR::ABL1, PML::RARA, CBFB::MYH11, KMT2A::AFF1) using cloud-based bioinformatics workflows with novel custom fusion finder software. We detected fusion genes in 100% of cell lines with the expected breakpoints and confirmed the presence or absence of a recurrent fusion gene in 12 of 14 patient cases. With our optimized assay and cloud-based bioinformatics workflow, these assays and analyses could be performed in under 8 hours. The platform's portability, potential for adaptation to lower-cost devices, and integrated cloud analysis make this assay a candidate to be placed in settings like LMIC to bridge the need of bedside rapid molecular diagnostics.https://doi.org/10.1371/journal.pgph.0002267 |
| spellingShingle | Olga Sala-Torra Shishir Reddy Ling-Hong Hung Lan Beppu David Wu Jerald Radich Ka Yee Yeung Cecilia C S Yeung Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing. PLOS Global Public Health |
| title | Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing. |
| title_full | Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing. |
| title_fullStr | Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing. |
| title_full_unstemmed | Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing. |
| title_short | Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing. |
| title_sort | rapid detection of myeloid neoplasm fusions using single molecule long read sequencing |
| url | https://doi.org/10.1371/journal.pgph.0002267 |
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