Document Type

Article

Publication Date

9-2-2026

Identifier

DOI: 10.1371/journal.pcbi.1014423; PMCID: PMC13585337

Abstract

Microsatellite instability (MSI) is a key biomarker of mismatch repair deficiency and response to immunotherapy, yet most existing genomic detection methods are optimized for short-read sequencing and rely on a panel of homopolymer markers, limiting the ability to characterize genome-wide and motif-specific patterns of instability. Here we present Owl, a bioinformatic tool for quantifying MSI from long-read (PacBio) genomic data. Owl leverages a genome-wide marker set of more than 140,000 microsatellite repeats ranging from 1-6 bp in length to measure MSI across a phased genome. Using a wrap-around alignment algorithm, Owl constructs repeat-length distributions at each marker site and flags somatic instability using the coefficient of variation. We applied Owl to screen for markers with stable coverage, phasing, and baseline variation across 131 diverse genomes from the Human Pangenome Reference Consortium, where Owl scores ranged from 1.4% to 5.4% of markers exceeding the instability threshold. When applied to cancer cell lines and one diffuse astrocytoma tumor-normal pair, Owl identified six MSI genomes with 10-27% unstable markers and showed close concordance with an Illumina DRAGEN MSI assay for the astrocytoma sample. Motif-level analyses revealed shared enrichment of short homopolymer and dinucleotide (A- and AT-rich) repeats across MSI cancers. Owl is implemented in Rust and integrated into the PacBio HiFi Somatic workflow, providing a scalable framework for MSI analysis from long-read sequencing focused on repeat instability specifically in tumor samples.

Journal Title

PLoS Comput Biol

Volume

22

Issue

9

First Page

1014423

Last Page

1014423

MeSH Keywords

Microsatellite Instability; Humans; Computational Biology; Algorithms; Microsatellite Repeats; Genomics; Sequence Analysis, DNA; Software; Genome, Human; Cell Line, Tumor

PubMed ID

42685296

Keywords

Microsatellite Instability; Computational Biology; Algorithms; Microsatellite Repeats; Genomics; DNA Sequence Analysis; Software; Human Genome; Tumor Cell Line

Comments

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Publisher's Link: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1014423

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