Hybrid Sequencing to Define the Full-Length Transcriptome of Double Stranded DNA Viruses

 描述(申请人提供)：最近的研究表明，大的双链DNA(DsDNA)病毒蛋白质组比以前认识的要复杂得多。核糖体图谱和质谱分析已经在多种dsDNA病毒中发现了数百个新的编码区，这引发了dsDNA病毒如何编码和调节这种膨胀的蛋白质组的根本问题。我们对dsDNA病毒产生如此多样化的蛋白质组的机制了解有限，这在很大程度上是因为我们对病毒转录本结构的了解很少。确定全长病毒转录本的序列 (全长转录组)识别编码病毒蛋白的mRNAs，以及潜在的编码目前未被识别的病毒蛋白的新转录本。定义全长转录本还提供了对病毒启动子和调节mRNA稳定性和翻译效率的5‘非翻译区(UTRs)的全局看法。标准的RNA-Seq方法不足以识别全长dsDNA病毒转录本，因为病毒基因组的广泛转录产生了重叠的转录单位，这可以防止转录末端的定义，并使剪接连接到特定病毒转录本的分配复杂化。因此，还没有为任何大型dsDNA病毒定义全长转录组。需要新的、高通量的方法来定义全长dsDNA病毒转录本，从而了解病毒基因表达的调节和驱动病毒蛋白质组复杂性的机制。以人巨细胞病毒(HCMV)为模型dsDNA病毒，我们提出了一种创新的技术和分析相结合的方法来定义全长dsDNA病毒转录本。我们的多学科方法使用新的生物信息学方法来合并长短读取长度测序数据，即所谓的混合测序，与高清晰度转录起始点分析相结合，以提供全基因组范围内全长病毒转录本的端到端视图。我们的初步结果表明，我们的方法识别了由选择性转录起始使用和剪接产生的新的HCMV编码区，以及控制病毒转录本表达的新启动子。这个项目的成功完成将为确定dsDNA病毒全长转录本提供一个新的范例，这将有助于确定驱动dsDNA病毒蛋白质组复杂性的机制，以及控制病毒基因表达的调控机制。

 DESCRIPTION (provided by applicant): Recent studies show that large double stranded DNA (dsDNA) virus proteomes are much more complex than previously appreciated. Ribosome profiling and mass spectrometry analyses have identified hundreds of novel coding regions in multiple dsDNA viruses, raising the fundamental question of how do dsDNA viruses encode and regulate such expansive proteomes? We have a limited understanding of the mechanisms by which dsDNA viruses generate such diverse proteomes, in large part due to our poor understanding of viral transcript structure. Defining the sequence of full-length viral transcripts (the full-length transcriptome) identifies the mRNAs that encode viral proteins, and potentially novel transcripts encoding currently unrecognized viral proteins. Defining full-length transcriptomes also provides a global view of viral promoters, and 5' untranslated regions (UTRs) that regulate mRNA stability and translation efficiency. Standard RNA-Seq approaches are insufficient to identify full-length dsDNA virus transcriptomes as widespread transcription of the viral genomes creates over-lapping transcriptional units that can prevent the definition of transcript termini, and complicate assignment of splice junctions to specific viral transcripts. Asa result, the full-length transcriptome has not been defined for any large dsDNA virus. New, high throughput approaches are needed to define full-length dsDNA virus transcriptomes, and thereby understand the regulation of viral gene expression and the mechanisms driving viral proteome complexity. Using human cytomegalovirus (HCMV) as a model large dsDNA virus, we propose an innovative combination of techniques and analyses to define full-length dsDNA virus transcriptomes. Our multi-disciplinary approach uses novel bioinformatics methods to merge long and short read length sequencing data, known as hybrid sequencing, with high definition transcription start site analysis to provide an "end-to-end" view of full-length viral transcripts n a genome-wide scale. Our preliminary results show that our approach identifies novel HCMV coding regions resulting from alterative transcription start usage and splicing, and new promoters controlling the expression of viral transcripts. Successful completion of this project will provide a new paradigm for defining full-length dsDNA virus transcriptomes that will be useful for determining the mechanism driving dsDNA virus proteome complexity, and the regulatory mechanisms controlling viral gene expression.