Improved Library Preparation Workflows for Next Generation Sequencing

描述（由申请人提供）：下一代测序（NGS）技术是一个快速发展的领域，能够以较低的成本产生千兆比特的测序数据。虽然NGS在基因组DNA测序（DNA-Seq）方面得到了最好的认可，但仍有大量工作要扩大正在研究的样本类型的广度。RNA深度测序（RNA-Seq）是NGS实验的一个成熟部分，越来越关注小RNA深度测序（smRNA-Seq）。20-30 nt小RNA的深度测序的限制之一是样品制备工作流程，其需要几个纯化步骤。与所有RNA样品一样，NGS样品制备涉及将固定序列（称为接头）连接到起始RNA文库的5 <$端和3 <$端。该连接步骤可能受到衔接子序列彼此之间没有文库区段的不期望的连接的困扰，导致衔接子二聚体形成。虽然在大多数文库制备工作流程中可以使用亲和捕获来去除衔接子二聚体，但是衔接子标记的小RNA文库在尺寸上太接近于衔接子二聚体，从而无法有效使用该方法。因此，需要凝胶纯化步骤，这可能耗尽低丰度序列。为了避免无意中改变文库的复杂性，有利的是在连接步骤阻断衔接子二聚体形成。考虑到这一目标，我们提出了一种新的方法来制备NGS的RNA样品，该方法利用TriLink在核酸化学方面的专业知识来反对衔接子-衔接子连接，同时允许有效连接 在文库的5 '和3'末端上的接头探针。

DESCRIPTION (provided by applicant): Next generation sequencing (NGS) technologies are a rapidly developing area, with the ability to produce gigabases of sequencing data at reduced costs. While NGS is best recognized for genomic DNA sequencing (DNA-Seq), there is a significant body of work to expand the breadth of sample types being studied. RNA deep sequencing (RNA-Seq) is a well-developed segment of NGS experimentation, with a growing focus on small RNA deep sequencing (smRNA-Seq). One of the limitations in the deep sequencing of the 20-30 nt small RNA is in the sample preparation workflow, which requires several purification steps. As with all RNA samples, NGS sample preparation involves the ligation of fixed sequences, called adapters, onto the 5¿ and 3¿ ends of the starting RNA library. This ligation step can be plagued by the undesired joining of the adapter sequences to one another without a segment of the library in between, resulting in adapter dimer formation. Although affinity capture can be used to remove adapter dimers in most library prep workflows, adapter-tagged small RNA libraries are too close in size to the adapter dimers for effective use of this approach. As result, a gel purification step is required, which can deplete low abundance sequences. To avoid making unintentional changes to the complexity of the library, it is advantageous to block adapter dimer formation at the ligation step. With this goal in mind, we propose a novel approach to RNA sample preparation for NGS that uses TriLink's expertise in nucleic acid chemistry to disfavor adapter-adapter ligation while allowing for efficient joining of adapter probes on the 5'and 3' ends of the library.