High Fidelity Transcriptome Sequencing Tools

描述(申请人提供)：基因表达调控驱动所有生物过程。彻底了解支配基因表达模式的机制对于了解生物学的基本原理以及癌症和其他疾病的复杂性是必要的。人类疾病研究的未来将基于人类基因组和转录组的下一代测序(NGS)；并将依赖于DNA和RNA文库的准确序列表示。近年来，随着NGS的出现，DNA测序的成本大幅下降，这彻底改变了基因表达的研究方式。在这个新的范例中，转录组被一种被称为逆转录酶的酶转化为互补DNA(CDNAs)。核苷酸序列分析揭示了mRNAs的遗传密码以及许多转录后过程步骤，这些步骤只能通过RNA测序来研究。这提供了所有表达的基因的综合视图，以及通过传统方法无法获得的额外信息。NGS平台越来越强大和可靠；然而，这些技术在转录组测序中的适应性还不太发达。作为转录组测序的关键步骤，所有RNA物种(转录本)到DNA(在NGS系统中分析的材料)的转换存在技术困难，并在低保真逆转录酶的转换过程中受到复制错误、重排和偏见的影响。这一转换过程非常耗时，并且容易发生操作员错误。通过长达十年的研究温泉病毒和噬菌体遗传学的计划，Lucigen发现并开发了一种名为PyroScrip RT的独特酶，与传统酶相比，该酶有望将RNA序列分析的准确性提高两个数量级，并大大减少重排频率。PyroScrip RT的高温优化也有望减少由于二级结构而产生的偏置。我们打算使用这种酶来开发和验证转录体文库合成试剂盒，该试剂盒结合了一种单一的、新颖的、高保真的聚合酶，用于RNA的逆转录和随后的DNA扩增。为了实现这一目标，我们将开发一种简单的cDNA文库合成协议，该协议对所有主要的NGS平台都是灵活的，并基于PyroScrip RT的独特性质，将减少误结合伪影和偏见。我们建议的研究结果将是一种试剂，将允许小型和大型研究实验室在平等的基础上竞争，并通过更准确和公正的序列数据库促进转录组多样性的知识。 公共卫生相关性：人类基因组和转录组(转录的RNA)包含与人类疾病相关的信息。随着下一代测序和改进的生物信息学分析技术的出现，我们能够更快速地表征疾病样本，从而增加我们对遗传性疾病的易感性和发病、引发癌症的体细胞突变以及影响人类的致病微生物的身份的了解。需要新的工具来提高准确性，消除重排，减少转录组研究的偏见，以及简化试剂盒，使所有研究人员都能轻松获得下一代测序。为这个项目提供的资金将帮助我们开发这些工具，并帮助加快人类疾病的研究。

DESCRIPTION (provided by applicant): The regulation of gene expression drives all biological processes. A thorough understanding of the mechanisms governing the patterns of gene expression is necessary to understand the fundamentals of biology and the complexities of cancers and other diseases. The future of research into human disease will be based in next generation sequencing (NGS) of the human genome and transcriptome (the set of all RNA molecules, including mRNA, rRNA, tRNA, and non-coding RNA produced in one or a population of cells); and will be dependent on accurate sequence representation from DNA and RNA libraries. The cost of DNA sequencing has dropped dramatically in recent years with the advent of NGS and this has revolutionized the way that gene expression is studied. In this new paradigm, the transcriptome is converted to complimentary DNA (cDNA) by a class of enzyme known as reverse transcriptase. Nucleotide sequence analysis reveals the genetic code for mRNAs as well as numerous post transcriptional process steps that can only be studied by RNA sequencing. This provides a comprehensive view of all expressed genes, as well as additional information not available through traditional approaches. NGS platforms are increasingly robust and reliable; however, the adaption of these technologies for transcriptome sequencing is less developed. A key step in transcriptome sequencing, the conversion of all RNA species (the transcripts) to DNA (the material analyzed in the NGS systems) is laden with technical difficulties and compromised by copying errors, rearrangements and biases during the conversion by low fidelity reverse transcriptases. This conversion process is time consuming and prone to operator error. Through a decade-long program to study the genetics of viruses and phage in hot springs, Lucigen has discovered and developed a unique enzyme called PyroScript RT that promises to improve the accuracy of RNA sequence analysis by two orders of magnitude compared to conventional enzymes and greatly reduce the frequency of rearrangements. The high temperature optimum of PyroScript RT also promises to reduce bias due to secondary structures. We intend to use this enzyme to develop and validate transcriptome library synthesis kits that incorporate a single, novel, high fidelity polymerase for reverse transcription of RNA and subsequent DNA amplification. To achieve this goal we will develop a simple protocol for cDNA library synthesis that is flexible for all of the major NGS platforms and based on the unique qualities of PyroScript RT that will reduce misincorporation artifacts and bias. The result of our proposed study will be a reagent that will allow both the small and large research laboratory to compete on equal grounds and advance the knowledge of transcriptome diversity through a more accurate and unbiased sequence database. PUBLIC HEALTH RELEVANCE: The human genome and transcriptome (transcribed RNA) contain information relevant to human disease. With the advent of next-generation sequencing and improved bioinformatics analysis techniques, we are able to more rapidly characterize disease samples and thus increase our understanding of the susceptibility and onset of inherited diseases, the somatic mutations that initiate cancers and the identity of pathogenic microbes that affect humans. New tools are needed to improve the accuracy, eliminate rearrangements and reduce the bias of transcriptomics research as well as simplified kits that provide easy access to next-generation sequencing to all researchers. The funding provided for this project will help us develop these tools and help speed the research on human diseases.