Small RNA Barcoded Sequencing

描述(申请人提供)：表达的变化已被证明与各种正常的生理过程以及包括癌症在内的疾病有关。研究已经表明，miRNAs可能为疾病诊断和预后分析的发展提供有用的标记物。目前，还没有基于发现的、快速多路复用的方法可以同时测量几个样本或肿瘤组织中miRNA的表达。下一代测序技术原则上非常适合对小的非编码RNA进行高通量测序。尽管有这样的能力，对大量小RNA样本进行测序仍然是耗时和昂贵的。我们提出了一种多重策略的发展，通过使用样本特定的短识别核苷酸序列来索引来同时对大量的小RNA进行排序。我们建议克服的挑战包括酶序列偏差，它可能更喜欢某些转录本，以及不同深度的索引对小RNA图谱的影响程度。索引将具有测量碱基错误率的优势，允许用户进行跨基因组研究、时间进程、药物诱导的细胞实验和监测样本之间的日常表达变异性。该项目的目标是创建一种试剂盒，用于制作用于大规模并行(下一代)测序的最小偏差、高度索引的小RNA文库，其构建方式使相同的文库能够容易地被实时聚合酶链式反应(QPCR)询问。 公共卫生相关性：最近，人们对将下一代测序技术应用于临床非常感兴趣。已有文献证明，microRNAs在恶性组织和细胞系中作为癌基因或肿瘤抑制因子发挥作用，并被证明在几种癌症的进展中具有潜在的生物标志物的作用。通过开发一种用于大规模并行小RNA测序的高效且无偏倚的条形码方法，现在将可以获得这些序列在组织中的独特轮廓。

DESCRIPTION (provided by applicant): Changes in expression have been shown to be associated with a variety of normal physiological processes as well as diseases including cancer. Studies have already shown that miRNAs may provide useful markers for the development of disease diagnostic and prognostic assays. Currently there are no discovery based, rapid-multiplexed methods available to simultaneously measure miRNA expression in several samples or tumor tissues. Next generation sequencing technologies are in principle very well suited for high- throughput sequencing of small non-coding RNAs. Despite this capacity, it is still time consuming and expensive to sequence large numbers of small RNA samples. We propose the development of a multiplex strategy to simultaneous sequence large numbers of small RNAs by indexing using sample-specific short identifying nucleotide sequences. Challenges that we propose to overcome include enzymatic sequence biases that may prefer certain transcripts over others and the extent to which indexing at different depths affects small RNA profiles. Indexing will has the advantage of measuring base error rate, allows the user to perform cross genomic studies, time courses, drug induced cellular experiments and monitor day to day expression variability between samples. The goal of this project is to create a kit for making minimally-biased, highly indexed small RNA libraries for massively parallel ("next-generation") sequencing, that are constructed in such a way to allow the same libraries to be easily interrogated by real-time PCR (qPCR). PUBLIC HEALTH RELEVANCE: Recently there has been much interest in applying next-generation sequencing technologies for clinical use. microRNAs have been documented to behave as oncogenes or tumor suppressors in malignant tissues and cell lines, and have been shown to have potential for use as biomarkers in the progression of several cancers. By developing an efficient and non-biased barcoding method for massively parallel small RNA sequencing, unique profiles of these sequences in tissues will now be attainable.