Hartley Stephen W, Mullikin James C, Klein David C, Park Morgan, Coon Steven L
Comparative Genomics Analysis Unit, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, 20892, United States of America.
Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892, United States of America.
PLoS One. 2016 Sep 29;11(9):e0163590. doi: 10.1371/journal.pone.0163590. eCollection 2016.
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.
可变异构体调控（AIR）极大地增加了转录组的多样性，并在众多生物过程和病理过程中发挥重要作用。然而，异构体水平差异调控的检测和分析具有难度，尤其是面对复杂且注释不完整的转录组时。在此，我们利用Illumina短读长/高通量RNA测序来鉴定55个在松果体中表现出神经调节性AIR的基因，然后使用另外两个互补的实验平台进一步研究和表征与巴德-比埃尔综合征和非综合征性视网膜色素变性相关的Ttc8基因。使用JunctionSeq分析工具在该基因中检测到了几个新外显子和剪接接头，包括两个新的可变转录起始位点，在几个独立实验中发现它们表现出不成比例的强神经调节性差异表达。这些高通量测序结果通过靶向定量PCR和长读长太平洋生物科学公司的单分子实时测序进行了验证和补充。我们证实了众多新剪接接头的存在以及两个新起始位点的选择性上调。此外，我们鉴定出了20多种在该组织中共表达的Ttc8基因新异构体。通过使用来自多个独立平台的信息，我们不仅大大降低了影响结果的错误、偏差和假象的风险，而且能够比通过任何单个平台更深入、更精确地表征Ttc8基因的调控和剪接。本文概述的混合方法是转录组研究中的一种强大策略。
