OMICs AND AI APPROACHES FOR MUSCLE DISEASES: O.06 Haplotype and variant phasing of large muscle genes with linked-read sequencing

Abstract

Molecular genetic analysis of large genes is demanding although high-throughput sequencing (HTS) has facilitated the task immensely. Ultra-rare or novel missense variants in large genes such as nebulin and titin are very common in the general population, which makes variant interpretation difficult in sporadic cases. The interpretation of rare heterozygous variants is especially difficult when samples from family members are lacking and thus, segregation analysis is not possible. Short-read HTS collapses a diploid genome to a single sequence, but the linked-read technology provides phase information of continuous DNA fragments and resolves the haplotypes of patients. Long-range information is achieved by linking the barcoded short-read sequences to each other in the data-analysis step, allowing multiple variants in the same gene to be phased. Link-read technology enables the identification of complex structural variation and repeat expansions using the HTS data. Linked-read whole exome (WES) and whole genome sequencing (WGS) have the potential to improve the sequencing quality of genes harboring segmental duplications, such as nebulin and titin. We have linked-read WES and WGS data of 30 individuals. For this study, we analyzed 10 genes, with the longest coding regions in the human genome associated with skeletal muscle disorders. Our aim was to investigate how well the linked-read technology succeeded to detect phase variants in these large genes, and whether this facilitated variant classification by adding crucial information. We also discuss the technical limitations of the method, such as quality standards of DNA samples, which need to be considered to obtain the best possible results. Our preliminary results indicate that the linked-read technology is likely to improve the diagnostics of neuromuscular disorders.