P.295Linked-read whole genome sequencing in patients with congenital myopathy

Abstract

Over the past decade, Next Generation Sequencing (NGS) methods have revolutionised the field of genetics. Currently, the usage of Whole Genome Sequencing (WGS) in addition to Whole Exome Sequencing (WES) and panel sequencing is increasing in research and in clinical diagnostics. In this study, we wanted to test whether a new NGS application — the Linked-Read (LR) library preparation — can improve the identification of disease-causing variants in patients with congenital myopathy. For LR library preparation, gDNA molecules are encapsulated inside emulsion droplets. Every microreaction provides a unique barcode to each of the DNA fragments inside the droplet. In the data analysis step, the barcoded short-read sequences are linked up to each other, providing long-range information. In addition to regular WGS information, this enables the identification of structural variants such as insertions or translocations. It also resolves the haplotypes of the patient. Moreover, LRs have the potential to improve the short-read alignment of repetitive regions in genes such as nebulin and titin. We selected three patients with a congenital myopathy for this pilot study. The prior analysis using targeted Sanger sequencing of candidate genes, a custom neuromuscular gene panel or WES had failed to identify the genetic cause of their disorder. Furthermore, all patient samples had previously been analysed for Copy Number Variation (CNV) using a Comparative Genomic Hybridisation array (CGH-array) for neuromuscular disorders designed by our group, but the molecular diagnosis had remained unresolved. Our preliminary results indicate that LR NGS library preparation method adds valuable information, especially of structural variation, and is likely to improve the diagnostics of neuromuscular disorders.