Premium
Development of a next‐generation sequencing platform for retinal dystrophies, with LCA and RP as proof of concept
Author(s) -
COPPIETERS F
Publication year - 2010
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2010.2161.x
Subject(s) - retinitis pigmentosa , sanger sequencing , dna sequencing , computational biology , indel , genetics , amplicon , biology , massive parallel sequencing , personal genomics , gene , genome , bioinformatics , human genome , single nucleotide polymorphism , polymerase chain reaction , genotype
Abstract Purpose Retinal dystrophies represent an emerging group of hereditary disorders that lead to degeneration of the photoreceptors and/or the retinal pigment epithelium, resulting in irreversible blindness. They are genetically complex, with over 200 disease loci identified so far. Current genetic screening consists of microarray analysis (Asper Ophthalmics) for the most recurrent mutations, and subsequent Sanger sequencing. However, the high cost and low throughput of the latter technology limits testing to only the most recurrent genes. This project aims to develop a high throughput and cost‐effective platform for screening of all known disease genes for Leber Congenital Amaurosis (LCA) and retinitis pigmentosa (RP), using the next‐generation sequencing (NGS) technology. Methods A NGS panel will be developed for all 16 and 47 known LCA and RP genes, respectively, including coding and untranslated regions, regulatory regions and microRNA binding sites. The protocol will consist of the following steps: 1) high throughput primerdesign and qPCR, 2) ligation, 3) shearing and 4) sequencing on the Illumina Genome Analyser IIx (GAIIx). This innovative protocol overcomes the need for short amplicons in order to render short‐read sequences by the GAIIx. This sequencing instrument was chosen because of its high capacity, low cost per base and the absence of interpretation problems at homopolymeric regions. Analysis of the variants will be performed using in‐house developed and commercial software, which ranks all variants according to their pathogenic potential. Conclusion Using the proposed protocol, comprehensive screening for all known disease genes for LCA and RP will be available for the first time.