Alternative Isoform Analysis of Ttc8 Expression in the Rat Pineal Gland Using a Multi-Platform Sequencing Approach...

Alternative isoform regulation (AIR) vastly increases transcriptome diversity and plays an important role in numerous biological processes and pathologies. However, the detection and analysis of isoform-level differential regulation is difficult, particularly in the face of complex and incompletely-annotated transcriptomes. Here we have used Illumina short-read/high-throughput RNA-Seq to identify 55 genes that exhibit neurally-regulated AIR in the pineal gland, and then used two other complementary experimental platforms to further study and characterize the Ttc8 gene, which is involved in Bardet-Biedl syndrome and non-syndromic retinitis pigmentosa. Use of the JunctionSeq analysis tool led to the detection of several novel exons and splice junctions in this gene, including two novel alternative transcription start sites which were found to display disproportionately strong neurally-regulated differential expression in several independent experiments. These high-throughput sequencing results were validated and augmented via targeted qPCR and long-read Pacific Biosciences SMRT sequencing. We confirmed the existence of numerous novel splice junctions and the selective upregulation of the two novel start sites. In addition, we identified more than 20 novel isoforms of the Ttc8 gene that are co-expressed in this tissue. By using information from multiple independent platforms we not only greatly reduce the risk of errors, biases, and artifacts influencing our results, we also are able to characterize the regulation and splicing of the Ttc8 gene more deeply and more precisely than would be possible via any single platform. The hybrid method outlined here represents a powerful strategy in the study of the transcriptome.