Directed evolution of RNA ligases for high-throughput sequencing

描述(由申请人提供)：虽然对小RNA的NextGen测序将提供对各种生物过程的重要见解，但测序过程固有地存在偏见，要么是通过文库建设，要么是由于RNA化学，导致对RNA丰度的巨大误报。为了减轻克隆RNA群体所固有的偏见，我们建议进行T4 RNA连接酶的定向进化。我们将使用一种新的乳化法来进化序列和结构非特异性的连接酶。我们还将扩展传统的克隆方法，试图识别特定的小RNA亚群，例如那些含有5‘-三磷酸的RNA。我们将通过对准备好的混合RNA和自然样本进行NextGen测序，来确定我们的正常化尝试和我们的新克隆策略的相对有效性。通过开展这项工作，我们可能会及时克服现在才认识到的获取和解释小RNA的高通量序列数据的巨大障碍。 公共卫生相关性：新的高通量测序方法(NextGen测序)正在提供关于基因组和基因表达的丰富数据，其中许多数据与了解人类健康以及开发人类疾病的诊断和治疗方法高度相关。然而，这些强大的方法似乎并不能准确地表示细胞中可能存在哪些小RNA分子。这种倾斜可能导致解释错误或遗漏与健康和疾病相关的关键序列(例如来自病毒的小的表达序列)。为了确保转录RNA的准确覆盖，我们将设计和进化新的试剂来准备下一代测序的样本。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.