Microsatellite Instability Profiling With Msisensor2 in Whole-Exome Sequencing Data From Healthy Controls and Colorectal Cancer Patients

Microsatellite instability (MSI) is a molecular phenotype linked to defects in DNA mismatch repair (MMR) and serves as a biomarker in colorectal cancer (CRC). Computational MSI assessment using next-generation sequencing data enables characterisation of microsatellite instability without a dedicated MSI assay. This study evaluated MSIsensor2 scores in blood-derived whole-exome sequencing (WES) data from healthy individuals and two CRC patients. Whole-exome sequencing (WES) data from peripheral blood samples of 235 healthy Kazakh individuals were obtained from the DNA Biobank of the Laboratory of Genomic and Personalised Medicine, National Laboratory Astana, Nazarbayev University, Astana, Kazakhstan. Blood-derived WES data from two patients with CRC, designated BR110 and BR114, were also analysed. Sequencing libraries were prepared using Illumina workflows and sequenced on the NovaSeq 6000 platform. MSIsensor2 was employed to assess MSI scores and quantify the number of unstable microsatellite loci detected. Relationships among MSI scores, locus coverage, and sequencing data volume were examined descriptively. Of the 235 healthy individuals, 205 samples (87.2%) had MSI scores below 10%, while 30 samples (12.8%) had scores between 10% and 40% per study-specific classification. No sample exceeded 40%. The CRC-associated blood samples BR110 and BR114 demonstrated MSI scores of 5.43% and 7.81%, respectively. BR110 exhibited 10 unstable microsatellite sites among 184 covered sites, whereas BR114 had 26 among 333 covered sites. The higher score in BR114 corresponded with increased sequencing coverage and a greater number of evaluable loci. These findings indicate variability in computational MSI measurements across blood-derived WES datasets but do not establish tumour MSI status. MSIsensor2 produced MSI-associated measurements from blood-derived WES data in both healthy individuals and patients with CRC. However, MSI scores from blood sequencing do not necessarily reflect tumour status; therefore, determining the clinical relevance of these findings requires investigation using tumour-derived material, established MSI reference assays, and a larger clinical cohort.