While NextGen sequencing of small RNAs will provide important insights into a variety of biological processes the sequencing process is inherently biased, either by library construction or by RNA chemistry, leading to large misrepresentations of RNA abundance. To alleviate the biases inherent in cloning RNA populations, we propose to carry out the directed evolution of T4 RNA ligase. We will use a novel emulsion method to evolve ligases that are sequence and structure non-specific. We will also expand conventional cloning methods to try to identify particular sub-populations of small RNAs, such as those that contain 5'-triphosphates. We will determine the relative efficacy of both our normalization attempts and our new cloning strategies through NextGen sequencing of both prepared mixes of RNAs and natural samples. Be undertaking this work we will likely overcome in a timely fashion what is only now coming to be realized as a gargantuan hurdle to the acquisition and interpretation of high-throughput sequence data for small RNAs.
New, high-throughput sequencing methods (NextGen sequencing) is providing a wealth of data on genomes and gene expression, much of which is highly relevant to understanding human health and to developing diagnostics and therapeutics for human disease. However, it appears as though these powerful methods do not accurately represent which small RNA molecules may be present in a cell. Such skewing could lead to errors in interpretation or to missing critical sequences relevant to health and disease (such as small, expressed sequences from viruses). In order to ensure accurate coverage of transcribed RNAs, we will design and evolve new reagents for preparing samples for NextGen sequencing.
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