480P Comparative analysis of CRISPR/Cas9-targeted Nanopore long-read sequencing approaches in repeat expansion disorders

• Published in: Neuromuscular disorders : NMD Vol. 43; p. 104441
• Main Authors: Bonne, G., Benarroch, L., Boëlle, P., Labrèche, K., Madry, H., Mohand-Oumoussa, B., Eura, N., Nishino, I., Gourdon, G., Tomé, S.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.10.2024
• ISSN: 0960-8966
• DOI: 10.1016/j.nmd.2024.07.326

Over the past 30 years, more than 50 repeat expansion disorders have been identified, thanks to the exponential development of genetic tools and next-generation sequencing technologies. Long-read sequencing has emerged as a new milestone in the diagnosis of these disorders. Despite these major achievements, the comprehensive characterization of short tandem repeats in a pathological context remains challenging, primarily due to their inherent characteristics such as motif complexity, high GC content, and variable length. In this study, we implemented CRISPR/Cas9-based enrichment strategy combined to Oxford Nanopore (ONT) long-read sequencing. Our aim was to thoroughly characterize trinucleotide repeat expansion in two neuromuscular diseases: oculopharyngodistal myopathy (OPDM) and myotonic dystrophy type 1 (DM1). We conducted precise analyses of the OPDM and DM1 loci, determining repeat size, repeat length distribution, and DNA methylation. We achieved this by distinguishing normal and expanded alleles, using three different basecallers (Guppy, Bonito and Dorado). Our observations revealed miscalling of CGG and CTG repeats, with errors highly dependent on library preparation protocols (ONT R.9.4 vs R10.4 chemistries) and the chosen basecalling strategy. Our findings highlight the importance of the choice of basecalling strategies for the true characterization of repeated regions. Given our results, we propose guidelines for the CRISPR/Cas9-enrichment long-read sequencing strategy for repeat expansion diseases, which could be readily applicable in research but also in diagnostic settings.