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

 描述(申请人提供)：人类转录组明显比其同源基因组更复杂，这是由于数十万种可能的异构体、等位基因特定的表达问题、可变的RNA编辑变化以及跨越细胞类型、发育阶段和生理压力的差异表达模式。下一代测序(NGS)平台通过以低成本、高通量的格式提供海量数据，正在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，因为 大多数转录本的阅读技术和从头组装仅通过短读NGS技术就受到了影响。即使有一个高质量的参考人类基因组(这是父母等位基因的马赛克)，转录组测序和组装也是一个巨大的挑战。在整个mRNA上进行单倍体分析对于理解RNA编辑的全部范围是至关重要的，如果不诉诸于克隆DNA，则不容易实现这一点。显然，需要新的工具来弥合大规模并行短读测序技术和组装完整mRNA分子的需要之间的差距。这笔赠款的SBIR第一阶段建议开发短读NGS技术，以沿其整个长度精确地对mRNA进行测序，而不考虑其大小。这项技术将使复杂转录本的准确组装成为可能，而不需要使用基于Sanger测序的策略进行cdna克隆和引物行走。这些工具的开发可以实现令人望而生畏的转录本的从头测序，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，并使个人转录组重新测序变得容易。

 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.