Interrogation of Microsatellite Instability in Ewings Sarcoma

Microsatellite instability (MSI) is a key biomarker of mismatch repair deficiency and response to immunotherapy, but current detection methods are optimized for short-read sequencing and focus on small sets of homopolymer markers. Here we present Owl, a bioinformatic tool for quantifying MSI from PacBio HiFi whole-genome data using a genome-scale panel of simple repeats. Owl uses a wrap-around alignment algorithm to measure repeat lengths at more than 140,000 microsatellite loci and summarizes instability with the coefficient of variation in repeat length across phased haplotypes. We first construct and refine a long-read compatible marker set using 131 diverse Human Pangenome Reference Consortium genomes, removing loci that are frequently no-called or unstable in controls. In this panel, Owl scores for controls range from 1.4% to 5.4% of markers exceeding the instability threshold. Applied to 30 cancer cell lines and one diffuse astrocytoma tumor-normal pair, Owl identifies five MSI-high genomes with 15-18% unstable markers and shows close agreement with an Illumina DRAGEN MSI assay on the astrocytoma sample. Motif-level analyses reveal shared enrichment of short A- and AT-rich repeats across MSI-high cancers and uncover a distinct pattern of elevated GGAA-microsatellite instability in Ewing sarcoma cell lines, consistent with the known role of EWS::FLI1 at GGAA-rich regulatory elements. Owl is implemented in Rust, and integrated into the PacBio HiFi Somatic workflow, providing a scalable framework for MSI analysis from long-read sequencing and for discovering cancer-specific patterns of repeat instability.