Caitlin A. Loh1,2,3, Danielle A. Shields1,2,3, Adam Schwing1,2 and Gilad D. Evrony1,2 1Center for Human Genetics and Genomics, New York University Grossman School of Medicine, New York, New York 10016, USA; 2Department of Pediatrics, Department of Neuroscience & Physiology, Institute for Systems Genetics, Perlmutter Cancer Center, and Neuroscience Institute, New York University Grossman School of Medicine, New York, New York 10016, USA

↵3 These authors contributed equally to this work.

Corresponding author: gilad.evronynyulangone.org Abstract

Microsatellites are highly mutable sequences that can serve as markers for relationships among individuals or cells within a population. The accuracy and resolution of reconstructing these relationships depends on the fidelity of microsatellite profiling and the number of microsatellites profiled. However, current methods for targeted profiling of microsatellites incur significant “stutter” artifacts that interfere with accurate genotyping, and sequencing costs preclude whole-genome microsatellite profiling of a large number of samples. We developed a novel method for accurate and cost-effective targeted profiling of a panel of more than 150,000 microsatellites per sample, along with a computational tool for designing large-scale microsatellite panels. Our method addresses the greatest challenge for microsatellite profiling—“stutter” artifacts—with a low-temperature hybridization capture that significantly reduces these artifacts. We also developed a computational tool for accurate genotyping of the resulting microsatellite sequencing data that uses an ensemble approach integrating three microsatellite genotyping tools, which we optimize by analysis of de novo microsatellite mutations in human trios. Altogether, our suite of experimental and computational tools enables high-fidelity, large-scale profiling of microsatellites, which may find utility in diverse applications such as lineage tracing, population genetics, ecology, and forensics.

Received November 28, 2023. Accepted July 11, 2024.