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Partner-independent fusion gene detection by multiplexed CRISPR/Cas9 enrichment and long-read Nanopore sequencing

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Item Type:Preprint
Title:Partner-independent fusion gene detection by multiplexed CRISPR/Cas9 enrichment and long-read Nanopore sequencing
Creators Name:Stangl, C. and de Blank, S. and Renkens, I. and Verbeek, T. and Valle-Inclan, J.E. and González, R.C. and Henssen, A.G. and van Roosmalen, M.J. and Stam, R.W. and Voest, E.E. and Kloosterman, W.P. and van Haaften, G. and Monroe, G.
Abstract:Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment possibilities. The promiscuity of fusion genes with respect to partner choice and exact breakpoint-positions restricts their detection in the diagnostic setting, even for known and recurrent fusion gene configurations. To accurately identify these gene fusions in an unbiased manner, we developed FUDGE: a FUsion gene Detection assay from Gene Enrichment. FUDGE couples target-selected and strand-specific CRISPR/Cas9 activity for enrichment and detection of fusion gene drivers (e.g. BRAF, EWSR1, KMT2A/MLL) - without prior knowledge of fusion partner or breakpoint-location - to long-read Nanopore sequencing. FUDGE encompasses a dedicated bioinformatics approach (NanoFG) to detect fusion genes from Nanopore sequencing data. Our strategy is flexible with respect to target choice and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in a single sequencing run. We observe on average a 508 fold on-target enrichment and identify fusion breakpoints at nucleotide resolution - all within two days. We demonstrate that FUDGE effectively identifies fusion genes in cancer cell lines, tumor samples and on whole genome amplified DNA irrespective of partner gene or breakpoint-position in 100% of cases. Furthermore, we show that FUDGE is superior to routine diagnostic methods for fusion gene detection. In summary, we have developed a rapid and versatile fusion gene detection assay, providing an unparalleled opportunity for pan-cancer detection of fusion genes in routine diagnostics.
Keywords:Fusion Genes, Detection, Enrichment, Semi-Targeted, Nanopore Sequencing
Publisher:Cold Spring Harbor Laboratory Press
Article Number:807545
Date:22 October 2019
Official Publication:https://doi.org/10.1101/807545

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