Integrated biochemical and bioinformatic technologies for accurate transcriptome-wide full-length RNA assembly.

集成生物化学和生物信息技术，用于准确的转录组全长 RNA 组装。

• 批准号: 9119934
• 负责人: Colin Noel Dewey
• 金额: $ 0.5万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-20 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9119934
• 关键词: Affect; Alleles; Alternative Splicing; Animals; Behavioral; Benchmarking; Biochemical; Bioinformatics; Biology; Brain; Cataloging; Catalogs; Cells; Cellular biology; Chimera organism; Cloning; Code; Complementary DNA; Complex; Computer software; DNA; DNA Resequencing; Data; Data Analyses; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Enzymes; Epigenetic Process; Event; Exons; Face; Gene Expression Profile; Gene Expression Regulation; Generations; Genes; Genetic; Genome; Genomics; Goals; Grant; Haplotypes; Health; Human; Individual; Length; Libraries; Malignant Neoplasms; Maps; Messenger RNA; Methods; Modification; Natural regeneration; Neurologic; Nucleotides; Pattern; Phase; Physiological; Plants; Process; Protein Isoforms; Proteins; Protocols documentation; RNA; RNA Editing; RNA Sequences; RNA Splice Sites; RNA Splicing; Reading; Regulation; Research; Resort; Reverse Transcription; Sampling; Scientist; Sequence Analysis; Site; Small Business Innovation Research Grant; Staging; Stress; Technology; Time; Transcript; Universities; Variant; Walking; Wisconsin; base; cDNA Library; cell type; computerized data processing; computerized tools; cost; differential expression; genome sequencing; human reference genome; innovation; neurological pathology; new technology; next generation sequencing; novel; open source; programs; single molecule; success; tool; tool development; transcriptome sequencing; transcriptomics

 DESCRIPTION (provided by applicant): The human transcriptome is significantly more complex than its cognate genome, due to the hundreds of thousands of possible isoforms, allele-specific expression issues, variable RNA editing changes, and differential expression patterns spanning cell types, developmental stages, and physiological stresses. Next- generation sequencing (NGS) platforms are fundamentally altering genetic and genomic research by providing massive amounts of data in a low-cost, high-throughput format. The main drawback of existing technologies is the short sequence read lengths they produce (Illumina) or the high error rate (PacBio). Identifying single nucleotide variations is problematic with the long read technology and de novo assembly of most transcripts is compromised with short read NGS technologies alone. Even with a high quality reference human genome (which is a mosaic of the parental alleles), transcriptome sequencing and assembly is a significant challenge. Haplotyping across an entire mRNA is critical for understanding the full extent of RNA editing and is not readily achieved without resorting to cloned DNA. New tools that bridge the gap between massively parallel short read sequencing technologies and the need to assemble complete mRNA molecules are clearly needed. The SBIR Phase I of this grant proposes to develop the short read NGS technology to accurately sequence mRNAs along their entire length, regardless of size. This technology will enable the accurate assembly of complex transcriptomes, without cDNA cloning and primer walking using Sanger sequencing based strategies. The development of these tools could enable the de novo sequencing of daunting transcriptomes, reduce computational costs of transcriptome assembly significantly, produce more complete and accurate catalogs of RNA edited transcripts, and make personal transcriptome resequencing tractable.

 描述（由申请人提供）：由于数十万种可能的同种型、等位基因特异性表达问题、可变RNA编辑变化以及跨越细胞类型、发育阶段和生理应激的差异表达模式，人类转录组明显比其同源基因组复杂。下一代测序（NGS）平台通过以低成本、高通量格式提供大量数据而从根本上改变遗传和基因组研究。现有技术的主要缺点是它们产生的短序列读取长度（Illumina）或高错误率（PacBio）。识别单核苷酸变异是有问题的， 阅读技术和大部分转录物的从头组装仅用短阅读NGS技术就受到损害。即使有高质量的参考人类基因组（其是亲本等位基因的嵌合体），转录组测序和组装也是一个重大挑战。整个mRNA的单倍型分析对于理解RNA编辑的完整程度至关重要，并且如果不诉诸克隆DNA就不容易实现。显然需要新的工具来弥合大规模平行短读段测序技术与组装完整mRNA分子的需要之间的差距。SBIR第一阶段计划开发短读NGS技术，以准确地对mRNA的沿着全长进行测序，而不考虑其大小。该技术将能够精确组装复杂的转录组，而无需使用基于桑格测序的策略进行cDNA克隆和引物步移。这些工具的开发可以实现对令人生畏的转录组的从头测序，显著降低转录组组装的计算成本，产生更完整和准确的RNA编辑转录本目录，并使个人转录组重测序易于处理。