Veronika Scholz, Veronika Schönrock, Hannes Erdmann, Vitus Prokosch, Maria Schoedel, Manuela Almus, Mayra Sauer, Veronika Mayer, Eva Breithausen, Inga van Buren, Christine Dineiger, Madeline Golibrzuch, Minerva Montero-Hernández, Annette Lischka, Katja Eggermann, Caroline Heintz, Ariane Hallermayr, Teresa Neuhann, Elke Holinski-Feder, Angela Abicht, Anna Benet-Pagès, Morghan C Lucas
Hereditary ataxias, caused by expansions of short tandem repeats, are difficult to diagnose using traditional PCR and Southern blot methods, which struggle to detect complex repeat expansions and cannot assess repeat interruptions or methylation. We present an updated Clinical Nanopore Cas9-Targeted Sequencing (Clin-CATS) workflow for analyzing repeat expansions, now compatible with the Oxford Nanopore Technologies R10 flow cell. The workflow incorporates the ONT wf-human-variation Epi2Me workflow, including the Straglr tool to analyze basecalled reads, ensuring compatibility with past, current, and future sequencing chemistries. It expands the number of genes analyzed from 10 to 27 and introduces new gene panels for ataxia, myopathy, neurodegeneration, and ALS/motor neuron disease. Validated with Coriell reference and clinical samples, this method improves the analysis of pathogenic repeat expansions, providing deeper insights into repeat structures while addressing the limitations of traditional approaches. In this work, we also explored the use of multiplexing, Flongle flow cells, and single-gene targeting as alternatives to panel-based approaches in the Clin-CATS workflow, finding that only single-gene targeting provides compatibility and reliable performance.