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Eric M. Morrow, Dilber E. Gamsiz; Genome-wide Investigation Of Alternative Splicing In The Neural Retina Using High-throughput RNA-sequencing. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3312.
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In order to understand the complexity of gene expression in the neural retina, we conducted high throughput resequencing of the polyA-enriched RNA from the murine transcriptome of neural retina.
PolyA-enriched mRNA was prepared from the murine retina. Two independent libraries were sequenced extensively using the Illumina Genome Analyzer IIx. Each library was prepared from neural retinas pooled from two distinct litters from CD1 inbred mouse strain. Each library (EMA and EMB) were sequenced on two lanes on the GAIIx, totalling greater than 40 million, pair-end 75 base pair reads per experiment . Analysis was conducted using Bowtie 0.12.7 (Langmead et al., 2008) for sequence alignment to the mouse genome (Build MM9). Splice junctions were subsequently mapped using Tophat 1.1.0 (Trapnell et al., 2009) and AceView mRNA/EST reference library, release June 22, 2007 (Thierry-Mieg and Thierry-Mieg, 2007). Finally, the Cufflinks 0.9.1 tool (Trapnell et al., 2010) was used to quantitate transcript using fragments per kilobase of exon model per million mapped fragments (FPKM) estimates. The distinct libraries (EMA and EMB) were considered biologic replicates, and gene expression, including novel and known transcripts, were compared across these two samples.
Through comparison of sequencing in two distinct libraries, 15258 different known genes were found to be expressed in adult neural retina. The most abundantly expressed genes included well-known genes of the neural retina (such as rhodopsin, S-antigen and others); however, novel AceView transcripts were also noted among the most abundantly expressed genes. Gene expression varied from 0.125 to 2481.5 FPKM with an average expression level of 8.61 FPKM. Alternatively splicing was noted in 3665 genes and a total of 20558 transcripts were identified. 142 disease genes were annotated and appeared to be expressed in high abundance relative to non-disease genes. As well, disease genes appeared to demonstrate a high level of alternative splicing relative to non-disease genes. A sizable number of previously unknown transcripts are being curated across the two sequence samples.
High throughput RNA-sequencing using novel massively parralel sequencing methods have uncovered a number of critical new insights into the structure of the transcriptome in neural retina. This initial application of these technologies reveal important insights into gene expression and alternative splicing, and will serve as a critical reference for gene discovery and development of novel treatment targets in disease models.
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