Rapid detection of myeloid neoplasm fusions using Single Molecule Long-Read Sequencing

Recurrent gene fusions are common drivers of disease pathophysiology in leukemias. Identification of these structural variants helps stratify disease by risk and assists with therapy choice. Current fusion detection methods require long turnaround time (7-10 days) or advance knowledge of the genes involved in the fusions. To address the need for rapid identification of clinically actionable fusion genes in heme malignancies without a-priori knowledge of the genes, we describe a long-read sequencing DNA assay designed with CRISPR guides to select and enrich for recurrent leukemia fusion genes. 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-AF4) 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. Key pointsO_LIWe describe a CRISPR-Cas9 enrichment Nanopore sequencing assay with streamlined bioinformatics that outperforms other fusion detectors. C_LIO_LIWe successfully detected both fusion genes and specific breakpoints in CML, APL, and AML in under 8 hours in 80% of patients. C_LI Visual Abstract (Figure 1)O_LIWe successfully detected fusion genes in hematological malignancies with a fast and efficient long-read sequencing workflow in under 8 hours. The method makes the genomic characterization of BCR-ABL1 DNA breakpoint in patients quick and simple, which potentiates design of patient specific primers for personalized monitoring MRD assays. C_LIO_LIOur assay is based on a CRISPR-Cas9 non-amplification enrichment library preparation strategy and uses Nanopore sequencing single stranded genomic DNA coupled with streamlined bioinformatic workflow containing a novel fusion detector software which outperforms current fusion detection software. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/22276469v1_fig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1809e63org.highwire.dtl.DTLVardef@11eb3e6org.highwire.dtl.DTLVardef@b4f7fborg.highwire.dtl.DTLVardef@10496ab_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Chemistry and bioinformatics workflow of rapid single molecule long-read sequencing. Genomic DNA may contain a target fusion gene. The CRISPR-Cas9 system binds via specific guideRNA (gRNA) designed to enrich for DNA containing regions of interests. The library preparation does not undergo any amplification, and simply requires dA tailing and adapter ligation and a clean-up prior to being loaded onto a sequencing flow cell. On the flow cell libraries are sequenced by nanopores. Data from the sequencing devices are streamed onto biodepot workflow builder and are analyzed starting from FAST5 files, through an initial quality control, then base calling and alignment. After alignment different fusion finder tools were tested including LongGF and BFF before visualizing on IgV for confirmation and interpretation. C_FIG