miR-SEQ: Highly efficient targeted quantification of extracellular miRNAs by Next Generation Sequencing

摘要： 最近的发现强调了无细胞miRNAs（cf-miRNAs）作为有前途的诊断和治疗的重要性。 癌症和许多其他疾病的预后生物标志物。生物流体，如血浆，可以通过 与组织活检不同，侵入性最小这与cf-miRNAs在生物流体中的高稳定性一起， 使得它们与其他更不稳定的生物分子相比在分子诊断中的应用具有吸引力。然而，在这方面， 目前的技术不足以对生物流体中的miRNA进行灵敏、特异和可靠的定量。 微阵列和RT-qPCR目前是cf-miRNAs表达谱分析的首选工具，尽管 每一种都有主要的缺点。微阵列具有低灵敏度、低动态范围和不能在微阵列中检测的缺点。 区分密切相关的miRNA序列，而RT-qPCR具有有限的多重能力， 放大偏差虽然下一代测序（NGS）在许多方面都上级，但其 生物流体中cf-miRNA谱的可靠性由于偏向于特定的 miRNA序列，大量无关的测序数据，凝胶纯化的需要， 扩增子及其高成本。在这里，我们提出了一种构建cf-miRNA测序文库的新方法 来解决这些问题。被称为miR-SEQ，它结合了杂交和 酶促步骤，以简化miRNA测序文库的制备，同时显著降低 测序偏倚它涉及一种靶向测序方法，允许定量所有miRNA， 感兴趣的包括稀有组织（例如，癌症）衍生的miRNA种类及其代表最高的 作为生物标志物感兴趣，否则将由没有或低数量的测序读数表示， 使得它们的量化有问题且昂贵。在第一阶段，我们证明了我们的可行性， 通过靶向测序方法准确检测超过100种cf-miRNAs。在第二阶段 我们将彻底优化miR-SEQ，以最大限度地提高其灵敏度，并允许对更多种类的 cf-miRNA的商业可行性。此外，我们亦会精简议定书，以便在年底前通过。 用户包括学术实验室、合同研究机构、企业研发和分子诊断实验室。

Abstract: Recent discoveries highlight the importance of cell-free miRNAs (cf-miRNAs) as promising diagnostic and prognostic biomarkers for cancer and many other diseases. Biofluids such as plasma can be accessed with minimal invasiveness, unlike tissue biopsies. This, together with the high stability of cf-miRNAs in biofluids, makes them attractive for use in molecular diagnostics compared to other, more labile biomolecules. However, current techniques are inadequate for sensitive, specific and reliable quantification of miRNAs in biofluids. Microarrays and RT-qPCR are currently the preferred tools for expression profiling of cf-miRNAs, although each has major drawbacks. Microarrays suffer from low sensitivity, low dynamic range, and the inability to distinguish closely related miRNA sequences, while RT-qPCR has limited multiplexing capability and amplification biases. While next-generation sequencing (NGS) is superior in many of these respects, its reliability for cf-miRNA profiling in biofluids is limited due to bias (under- and over-detection) towards particular miRNA sequences, overwhelming amounts of unrelated sequencing data, the need for gel-purification of amplicons, and its high cost. Here we propose a new approach for constructing cf-miRNA sequencing libraries that addresses these problems. Called miR-SEQ, it incorporates a new combination of hybridization and enzymatic steps to simplify the preparation of miRNA sequencing libraries while significantly decreasing the sequencing bias. It involves a targeted sequencing approach allowing the quantification of all miRNAs of interest including rare tissue- (e.g., cancer-) derived miRNA species and their isomiRs representing the highest interest as biomarkers that would otherwise be represented by none or low numbers of sequencing reads, making their quantification problematic and expensive. In Phase I we demonstrated the feasibility of our approach by accurately detecting more than 100 cf-miRNAs with a targeted sequencing approach. In Phase II we will thoroughly optimize miR-SEQ to maximize its sensitivity and to allow sequencing of a larger variety of cf-miRNAs for commercial viability. In addition, we will streamline the protocol to facilitate its adoption by end users including academic labs, contract research facilities, corporate R&D and molecular diagnostic labs.