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Alex H. Wagner, Adam P. DeLuca, Thomas L. Casavant, Todd E. Scheetz, Edwin M. Stone, Robert Mullins, Terry A. Braun; RNA Sequencing for Identification of Genetic Factors in Retinal Disease. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4527.
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© ARVO (1962-2015); The Authors (2016-present)
Despite advances in human genome sequencing, the transcriptome of tissues involved in pathology is not well understood. Recent studies of the male germ cell associated kinase (MAK) showed that the human gene annotation reference lacked an important retina specific exon. In this study, we sought to identify novel exons expressed in retinal tissue. Additionally, this study ranks gene likelihood to be causative of disease in the human eye.
RNA was isolated from human macular retina, including the fovea centralis. The poly-A enriched RNA was Illumina sequenced to measure expression and identify novel retina-specific exons and transcripts. RNA sequences were mapped to a reference genome and exons were identified with TopHat/Cufflinks. These exons were compared to RefSeq annotation to identify novel exons specific to the retina. Candidate exons were selected by splice read support (on both ends) and a continuous ORF in the direction of the containing transcript. Phylogenetic conservation scores and tissue specificity were used to promote likely exons. Tissue specificity was determined by comparison of retina microarray expression profiles to other ocular tissues.
Using RNA-seq and bioinformatics we have predicted exons that are not annotated by RefSeq that are expressed in human retina. This process successfully identified a previously uncharacterized isoform of MAK containing a novel exon. Likely novel exons are ranked by: number of reads, read quality, expression (FPKM values), phylogenic conservation, localization to known disease genes and continuity of ORF.
Next-generation sequencing technology has facilitated unique opportunities to generate data that probes the transcriptome of the eye to unprecedented levels of resolution. RNA sequencing has the advantage over hybridization array platforms of being able to identify previously unannotated transcriptional events in eye tissues. Using this technology, we have identified novel retina-specific exons that warrant additional inspection for their role in the retina and disease.
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