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.
Highlights
Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment
We developed FUDGE to enrich for fusion genes in which only one gene partner is known and for which the other fusion gene partner and/or breakpoint is unknown
The crRNAs are designed in a strand-directed manner to direct reads upstream or downstream of the crRNA sequence — effectively sequencing into the suspected 5′ or 3′ fusion partner (Fig. 1b, Methods, and Supplementary Fig. 1)
Summary
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. We develop FUDGE (FUsion Detection from Gene Enrichment) to accurately and impartially identify fusions. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. Recent studies suggest that up to 16% of cancers are driven by a fusion gene[1]. One gene is a recurrent fusion partner (e.g., KMT2A/MLL, ALK) which exhibits a tissue-specific pattern[3]. These genes can fuse to a multitude of partners to obtain their oncogenic potential. The high levels of variability in fusion gene configurations drastically limits diagnostic detection
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