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Ralph J. Florijn, Renate C. Zekveld-Vroon, Arne Bakker, Martin A. Haagmans, Jaroslav Skokan, Mary J. van Schooneveld, Olaf R. Mook, Arthur A. Bergen; A Novel Strategy For Fast And Cost-effective Mutation Detection In Retinitis Pigmentosa Genes Using Next Generation Sequencing. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4518.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa can be caused by multiple genes, for example 28 genes in ARRP, 18 genes in ADRP or 8 genes in LCA. Next generation sequencing is the method of choice for sequencing all these genes simultaneously. In this project we designed and validated a next generation sequencing approach allowing flexible and cost effective analysis of multiple genes in multiple patients using a novel procedure. We developed a multiplex PCR strategy and combined this with the Roche 454 sequencing platform, which allows long read sequencing.
We designed a novel multiplex PCR method using barcodes, for nearly all known ARRP, ADRP and LCA genes. This strategy allows direct incorporation of barcodes and necessary linkers for next generation sequencing in the fragments. After pooling these barcoded fragments, they were sequenced by standard next generation sequencing procedures and analysed using the NEXTGENE (softgenetics) analysis program. This procedure can be easily optimized in respect of number of reads and distribution of the sequencing reads.
Our sequencing approach was validated by sequencing 60 known sequence changes derived from 52 different DNA samples in one experiment. In additional experiments (half 454 run) at least 3 patient samples were sequenced for respectively ARRP, ADRP or LCA genes. Coverage of at average 85 reads for each amplicon were obtained and up to 90 percent of the fragments were sequenced in the first round. In a second experiment, the procedure was optimized resulting in more even distribution of the reads and better distribution of the fragments. New mutations, such as nonsense mutations were identified in different genes and patients and will be presented.
We designed and validated a novel, cost effective and easy to optimize next generation sequencing strategy for mutation analysis in ARRP, ADRP or LCA and identified several new mutations. Because of the flexibility of this novel procedure, we can easily extend this approach to additional (new) disease genes or other multigene eye disorders.
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