Abstract
Obtaining reliable and high fidelity next-generation sequencing (NGS) data requires to choose a suitable sequencing platform and a library preparation approach, which both have their inherent assay-specific limitations. Here, we present the results of successful adaptation of SureSelect hybridisation-based target enrichment protocol for the sequencing on the Ion Torrent S5 platform, which is designed to work preferably with amplicon-based panels. In our study, we applied a custom NGS panel to screen a cohort of 16 unrelated patients affected by premature fusion of the cranial sutures, i.e. craniosynostosis (CS). CS occurs either as an isolated malformation or in a syndromic form, representing a genetically heterogeneous and clinically variable group of disorders. The approach presented here allowed us to achieve high quality NGS data and confirmed molecular diagnosis in 19% of cases, reaching the diagnostic yield similar to some of the published research reports. In conclusion, we demonstrated that an alternative enrichment strategy for library preparations can be successfully applied prior to sequencing on the Ion Torrent S5 platform. Also, we proved that the custom NGS panel designed by us represents a useful and effective tool in the molecular diagnostics of patients with CS.
Highlights
Routine next-generation sequencing (NGS) diagnostics requires high-quality sequencing data, short turnaround time and reasonable cost of the investigations
Using a cohort of patients presenting with craniosynostosis, we emphasise the utility of targeted gene panel sequencing in the diagnostics of this aetiologically heterogeneous condition
Since targeted NGS is regarded as a useful diagnostic method in identification of causative variants, especially in genetically heterogeneous diseases[23,24], we have designed and applied a custom hybridisation-based panel (Agilent Technologies) to screen CS patients with negative results of preliminary molecular screening
Summary
Routine NGS diagnostics requires high-quality sequencing data, short turnaround time and reasonable cost of the investigations. Successful implementation of targeted NGS in medical diagnostics results from several advantages It generates disease-restricted data with fewer variants of uncertain significance, simplifying the analysis. An alternative approach, i.e. hybridisation-based enrichment protocols such as SureSelect (Agilent Technologies) is available and is commonly applied on the Illumina platforms. Since targeted NGS is regarded as a useful diagnostic method in identification of causative variants, especially in genetically heterogeneous diseases[23,24], we have designed and applied a custom hybridisation-based panel (Agilent Technologies) to screen CS patients with negative results of preliminary molecular screening (involving hot-spot mutations located in exon 7 of FGFR1, exons 7 and 8 of FGFR2, and exon 7 of FGFR3, as well as we analysed the entire coding sequence of TWIST1)
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