Research Project
8054253
DESCRIPTION (provided by applicant): Methods for in-depth characterization of transcriptomes and quantification of transcript levels have emerged as valuable tools for understanding cellular physiology and human disease biology, and have begun to be utilized in various clinical diagnostic applications. Current methods, however, typically require RNA to be converted to cDNA prior to measurements. This step has been shown to introduce many biases and artifacts. In order to best characterize the "true" transcriptome, we propose the application of single molecule, true Direct RNA Sequencing (tDRS) in which RNA is sequenced without prior conversion to cDNA. The benefits of tDRS include the ability to use minute quantities (e.g. on the order of picograms) of RNA with minimal/no sample manipulation, the ability to analyze short RNAs which pose unique challenges for analysis using cDNA-based approaches, and the ability to perform these analyses in a low-cost and high-throughput manner. This application proposes strategies to adapt tDRS for multiple transcriptome analysis methods routinely used by the research and medical community. Combined with strategies for incremental improvements in read lengths, throughput and error rates, substantial progress will be made towards the ultimate goal of obtaining a bias-free view of transcriptomes. We will apply this technology to diverse RNA samples, including those from historical formalin-fixed, paraffin-embedded (FFPE) tissue specimens, leading to revolutionary advances in the understanding of biological and disease pathways. Our research plan brings together Helicos researchers and world experts in the field of RNA, genomics and medicine to achieve this goal. Our aims will be to: Specific Aim 1. Develop transcriptome analysis methods and tools for single-molecule Direct RNA sequencing of standard samples. Samples including yeast and ENCODE cell line RNAs will be analyzed with unprecedented detection and quantitation performance. We will develop: 1) prep-free gene expression, 2) whole transcriptome Direct RNA Sequencing, and 3) paired ends/paired reads with Direct RNA sequencing. Specific Aim 2. Develop tools to process RNA samples from FFPE tissue samples with single- molecule Direct RNA Sequencing, and apply these for the sequencing of influenza virus genomes from circa 1918. Short RNA species, which can not be satisfactorily analyzed with cDNA-based methodologies, can be analyzed with tDRS. Our goals include: 1) tailing and sequencing minute quantities of FFPE Tissue RNA, 2) development of FFPE tissue RNA quantification methods, 3) rRNA reduction from fragmented RNA samples, and 4) sequence analysis of archival influenza virus genomes from circa 1918 autopsy tissues. Specific Aim 3. Optimize chemical and enzymatic reagents for use with single-molecule direct RNA sequencing. Short-term and long-term improvements in tDRS performance will allow longer read lengths, higher throughput and reduced errors. We will improve tDRS by: 1) screening and synthesis of modified nucleotides for superior performance, and 2) screening and mutagenesis of polymerases for optimal behavior.
