SCIA: A fast and widely applicable pipeline for measuring expanded repeat instability

The expansion of short tandem repeats is a feature of over 60 different human diseases. Ongoing somatic instability throughout a patients lifetime can influence disease progression and has emerged as a therapeutic target. Understanding its mechanism is essential for the identification of both drug targets and therapeutic interventions. A major obstacle towards this translational goal has been to measure changes in repeat size distribution in a timely manner. To address this, here we present Single Clone-based Instability Assay (SCIA), a streamlined experimental design that saves weeks in assessing the effect of a gene knockout on repeat instability. The approach avoids bulk cultures and does not require a reporter cell line. It uses targeted long-read sequencing as a readout for repeat instability. We have validated the approach using FAN1, PMS1, and MLH1 knockouts in HEK293-derived cells. We provide a visualization software that generates delta plots, extracts the instability frequency, the bias towards expansion or contraction, and the average size of the changes. Using SCIA, we find that although FAN1 knockout clones showed increased frequency of expansions, the size of the expansions were smaller. This highlights the wealth of information that can be extracted and the potential for novel insights into the mechanism of repeat instability.