Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing.
Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing.
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DOI：
10.1371/journal.pgph.0002267
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发表时间：
2023
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复发性基因融合是白血病疾病病理生理学的常见驱动因素。识别这些结构变异有助于根据风险对疾病进行分层，并有助于治疗选择。鉴于分析的复杂性、训练有素的技术支持以及可靠的电力供应，中低收入国家（LMIC）的精确分子诊断具有挑战性。目前的融合检测方法需要很长的周转时间（7-10天）或预先了解融合中涉及的基因。最近的技术发展使测序成为可能，而不需要复杂的分子实验室，这可能使偏远地区和低收入环境能够获得分子诊断。我们描述了一种设计有CRISPR指导的长读段测序DNA测定，以选择和富集复发性白血病融合基因，其不需要存在异常的先验知识。通过应用基于纳米孔的快速测序技术，我们对基因组DNA的长片段进行了测序，并成功地检测了细胞系和原代标本中的融合基因（例如，BCR：：ABL 1，PML：：RARA，CBFB：：MYH 11，KMT 2A：：AFF 1）使用基于云的生物信息学工作流程和新型定制融合查找器软件。我们在100%具有预期断点的细胞系中检测到融合基因，并在14例患者病例中的12例中确认了复发性融合基因的存在或不存在。通过我们优化的检测和基于云的生物信息学工作流程，这些检测和分析可以在8小时内完成。该平台的便携性、适应低成本设备的潜力以及集成的云分析使该检测成为放置在LMIC等环境中的候选者，以满足床边快速分子诊断的需求。
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.