MSIanalyzer: Targeted Nanopore Sequencing Enables Single Nucleotide Resolution Analysis of Microsatellite Instability Diversity.

We present MSIanalyzer, a targeted sequencing-based pipeline profiling microsatellite instability (MSI) at single-nucleotide repeat-unit resolution, with base-level annotation of non-repeat interruptions where identifiable. Using Oxford Nanopore sequencing of the five Bethesda panel loci, we characterized repeat-unit distributions across mismatch repair (MMR)-deficient and MMR-proficient cell lines and primary human tissue samples. Operating on individual sequencing reads, MSIanalyzer captures repeat motifs while allowing interruptions and applies cluster-aware statistical tests for between-sample comparisons that account for read-level clustering within samples. Treating DNA isolated from primary human lymphocytes as a control, MSIanalyzer revealed pronounced locus-specific repeat contractions and broadened allelic distributions in MMR-deficient colorectal cancer cell lines (HCT15, HCT116), intermediate repeat-distribution shifts in the MMR-deficient prostate cancer cell line DU145, and stable profiles in MMR-proficient controls.

Across loci, multiple repeat-unit alleles were frequently supported within individual samples, highlighting substantial intra-sample heterogeneity. In a pilot analysis of clinical tumor-normal pairs, a majority of loci were classified as unstable in an MSI-H case but not in two MSS cases, in alignment with results from a commercial fluorescent PCR and capillary electrophoresis-based MSI assay. Mixtures of DNA prepared as a model of variable tumor content further demonstrated expected attenuation of MSI-associated signals with decreasing tumor fraction, while remaining detectable at multiple loci under low-tumor-content (10%) conditions. Together, these results demonstrate that MSIanalyzer detects read-level microsatellite diversity and enables quantitative, distribution-level characterization of MSI patterns, providing a foundation for mechanistic studies of repeat instability and future clinical validation.