Purchase this article with an account.
R. Chen, H. Wang, K. Zhang, R. A. Lewis, J. R. Lupski, G. Mardon, R. A. Gibbs; Disease Mutation Detection by Next Generation Sequencing. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6355.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We have explored the potential of applying the Next-generation high-throughput DNA sequencing (NGS) techniques to identify mutations for human eye diseases.
We are performing NGS on a set of ten unrelated Leber congenital amaurosis (LCA) families that are mapped to novel disease loci. Through homozygosity mapping, several novel LCA disease loci have been identified in our patient collection. However, the size of these candidate loci ranges from 10Mb to 100Mb, making PCR followed by Sanger sequencing strategy cost prohibitive. To circumvent this problem, based on the size of each locus, either targeted or whole exonome sequencing are conducted for these families with a combination of sequencing platforms, including Illumina and SOLiD.
Our preliminary data indicates that high sensitivity and specificity can be reached by targeted sequencing. More than 90% of the targeted exons can be captured and sequenced to sufficient depth. So far, we have successfully identified the causative mutation for the first family tested. Sequencing for the rest of the nine families are currently underway. Results on the sequencing of these families will be reported.
Therefore, NGS coupled with genetic mapping and DNA capture technology represents a powerful method for positional cloning and can be readily applied for all human genetic diseases.
This PDF is available to Subscribers Only