Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.

Leukemias are genetically heterogeneous, and knowing the type of genetic aberration is clinically important for progression prognosis and choice of treatment. The standard-of-care (SOC) methods in leukemia diagnostics are karyotyping, SNP-array and FISH. Optical genome mapping (OGM) has emerged as their replacement as it can detect different types of structural aberrations simultaneously. However, although OGM can additionally detect much smaller aberrations (500 bp vs. 5-10 Mb with karyotyping), its resolution is too low to define the location of aberrations between two labels and the breakpoints are disputable when labels are not distinct enough. Here, we test whether Cas9-directed long-read sequencing (LRS) can fill this gap. From an internal Bionano implementation study we selected ten OGM calls either located in low resolution areas or with disputable breakpoints that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION / GridION device. We could confirm all the OGM aberrations with Cas9-directed LRS and the actual breakpoints of the OGM calls were located between 0.2-5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. We show that Cas9-directed LRS can fill the gap of low resolution OGM areas thereby improving the prediction of clinical significance. We envisage that this technique can be an relevant secondary technique in diagnostic workflows including OGM.