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Xinjing Wang, Danyao Nie, Angela Turner, Keith Wetherby, Kerry Gotez, Alexandra Garafalo, Rebecca Parrish, Santa J Tumminia, Kory Johnson, Yang Fann; High-throughtput NGS of 60 Genes Involved in Inherited Corneal Disorders. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1023.
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To develop a next-gen sequencing (NGS) based platform to detect coding sequence alterations in causative and disease-related genes for inherited corneal disorders (CDs).
An assay to detect sequence alterations in genes involved in inherited CDs has been developed. In this assay, a primer library which targeted 1830 amplicons from 853 exons of 60 genes involved in corneal function and/or retinal development was designed to enrich the target genomic region. The RainDance PCR enrichment method was used to produce enriched targets and the PCR products were sequenced using NGS by the Illumina MiSeq. The NGS data were processed using CLC Bio software. A series of custom filters was developed and applied to screen for sequence variations in patient samples. The identified variations were independently validated by Sanger sequencing. A number of bioinformatic tools such as PolyPhen-2 and SIFT were used to further evaluate the potential pathogenic variations.
In this pilot study, we analyzed 18 samples from patients with CDs. Preliminary bioinformatics analysis indicated that this procedure was able to cover 99% of target sequence. Data mining identified previously reported mutations and novel pathogenic-likely variants in a variety of genes responsible for corneal dystrophies in 15 of the 18 patients.
These results indicate that this assay is capable of screening mutations in CD patients, especially in simplex cases with high sensitivity and efficiency, and has powerful potential in clinical applications.
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