Comprehensive and routine analysis of leukaemia samples
Nanopore sequencing rapidly and accurately identifies gene fusions
Compared to current methods for detecting gene fusions, such as real-time PCR and ﬂuorescence in situ hybridisation (FISH), which take days or even weeks to perform, nanopore technology offers the advantages of rapid sequencing with real-time data generation. With the aim of rapidly detecting and characterising a variety of oncogenic fusion events, including the BCR-ABL1 fusion (which is present in nearly all patients with chronic myeloid leukaemia [CML]), Jeck et al. developed a workﬂow based on a modified Anchored Multiplex PCR (AMP) method for library construction1. Initial proof-of-concept experiments in the K562 cell line using gene-specific primers against BCR exons 1 and 2, along with sequencing on the MinION, demonstrated the ability of this assay to precisely delineate the BCR-ABL1 fusion junction (Figure 1). Furthermore, real-time analysis enabled confident detection of the fusion within just 5 minutes, with the first fusion read being generated within five seconds.
Based on these promising results, Jeck et al. applied an expanded AMP-based assay, targeting an array of oncogenic fusions, to a number of haematological malignancy specimens. Various fusion events were detected including PML-RARA — the hallmark of acute promyelocytic leukaemia. Sensitivity to this critical fusion was 100% in clinical research samples, even with a 1:10 dilution specimen. When multiplexing four samples on a fresh flow cell, all fusions could be detected within 6 hours of sequencing.
Application of this assay to previously characterised libraries from formalin-fixed, paraffin-embedded (FFPE) sarcoma specimens detected a range of gene fusions with high specificity, demonstrating that the problem of fragmentation and lower DNA quality in FFPE specimens did not impede accurate fusion detection using nanopore sequencing. Furthermore, the fraction of MinION reads mapping to a given fusion was higher than that observed with traditional short-read sequencing technology in all but one case, which the authors suggest is likely due to longer read length. The authors concluded that:
‘…nanopore sequencing has great promise as a broad fusion detection platform in time critical settings or for laboratories with a low volume of specimens for which a (rapid) fusion assay is indicated’1.
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In a slightly different approach, Cumbo et al. compared FISH followed by Sanger sequencing to long-range template multiplex PCR and nanopore sequencing, to analyse BCR-ABL1 DNA fusions3. With a sequencing depth of 400x over the BCR region, the team observed concordance between the nanopore and Sanger sequencing results in all CML samples studied, stating that
‘…the very low costs, the ease of use, and the length of the reads (hundreds of kilobases), make MinION an ideal tool for target sequencing’3.
Researchers from the Fred Hutchinson Cancer Research Centre in Seattle, aimed to develop a single method allowing the detection and analysis of mutations in FMS-like tyrosine kinase 3 (FLT3), a tyrosine kinase receptor involved in haematopoietic cell proliferation, differentiation, and apoptosis4. Duplications in FLT3 are associated with aggressive acute myeloid leukaemia (AML). The quick workflows and real-time data acquisition afforded by nanopore sequencing offers a distinct advantage over traditional sequencing technologies. The researchers designed an RNA amplicon sequencing assay producing a 2,400 bp product covering welldefined hotspot regions in FLT3. MinION sequencing provided rapid acquisition of full-length reads, which allowed the reliable detection of internal tandem duplication mutations4.
These case studies are taken from the clinical white paper.
1. Jeck, W.R. et al. A nanopore sequencing–based assay for rapid detection of gene fusions. J Mol Diagn. S1525-1578(17)30630-X (2018).
2. Jeck, W.R. Nanopore sequencing and rapid fusion testing – a ‘killer app’ in molecular pathology. Presentation. Available at: https://nanoporetech.com/resource-centre/william-jeck-nanopore-sequencing-and-rapid-fusion-testing-killer-app-molecular [Accessed: 11 February 2019]
3. Cumbo, C. et al. Genomic BCR-ABL1 breakpoint characterization by a multi-strategy approach for “personalized monitoring” of residual disease in chronic myeloid leukemia patients. Oncotarget. 9(13):10978-10986 (2018).
4. Yeung, C. and Sala-Torra, O. Clinical applications for real-time sequencing in leukaemia. Presentation. Available at: https://nanoporetech. com/resource-centre/direct-rna-cdna-sequencinghuman-transcriptome. [Accessed: 07 February 2019]