Partner independent fusion gene detection by multiplexed CRISPR-Cas9 enrichment and long read nanopore sequencing
Christina Stangl, Sam de Blank, Ivo Renkens, Liset Westera, Tamara Verbeek, Jose Espejo Valle-Inclán, Rocío Chamorro González, Anton G. Henssen, Markus J. van Roosmalen, Ronald W. Stam, Emile E. Voest, Wigard P. Kloosterman, Gijs van Haaften, Glen R. Monroe
Abstract
Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment. Fusion gene partner choice and breakpoint-position promiscuity restricts diagnostic detection, even for known and recurrent configurations. Here, we develop FUDGE (FUsion Detection from Gene Enrichment) to accurately and impartially identify fusions. FUDGE couples target-selected and strand-specific CRISPR-Cas9 activity for fusion gene driver enrichment - without prior knowledge of fusion partner or breakpoint-location - to long read nanopore sequencing with the bioinformatics pipeline NanoFG. FUDGE has flexible target-loci choices and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in one sequencing run. We observe on-average 665 fold breakpoint-site enrichment and identify nucleotide resolution fusion breakpoints within 2 days. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. FUDGE is a rapid and versatile fusion detection assay for diagnostic pan-cancer fusion detection.