Improved Library Preparation Workflows for Next Generation Sequencing

改进下一代测序的文库制备工作流程

• 批准号: 8455912
• 负责人: Natasha Paul
• 金额: $ 13.28万
• 依托单位: TRILINK BIOTECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8455912
• 关键词: Affinity; Area; Chemistry; Complementary DNA; DNA Sequence; DNA Sequence Analysis; Data; Data Analyses; Ensure; Evaluation; Event; Gel; Genomics; Goals; Government; Investigation; Lead; Libraries; Ligase; Ligation; Marketing; MicroRNAs; Mind; Modeling; Modification; Nucleic Acids; One-Step dentin bonding system; Persons; Phase; Plague; Positioning Attribute; Preparation; Process; Protocols documentation; RNA; RNA Sequences; RNA library; Randomized; Reading; Relative (related person); Sampling; Sequence Analysis; Small RNA; System; Techniques; Technology; Testing; Time; combinatorial; cost; deep sequencing; dimer; improved; instrument; meetings; next generation sequencing; novel strategies; public health relevance; research study

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.

描述（由应用程序提供）：下一代测序（NGS）技术是一个快速发展的领域，能够以降低的成本生产测序数据的千兆以上。虽然NGS在基因组DNA测序（DNA-Seq）中最能识别，但仍有大量的工作来扩展正在研究的样本类型的广度。 RNA深度测序（RNA-Seq）是NGS实验的发达段，越来越关注小RNA深测序（SMRNA-SEQ）。样品制备工作流程中，20-30 nt小RNA的深度测序中的局限性之一是需要几个纯化步骤。与所有RNA样品一样，NGS样品制备涉及固定序列（称为衔接子）的连接到启动RNA库的5和3端。这种连接步骤可能会因适配器序列的不希望的连接而困扰，而没有库之间的一段，从而导致了适配器二聚体的形成。尽管亲和力捕获可用于在大多数库准备工作流程中删除适配器二聚体，但适配器标记的小RNA库的大小太近，无法有效地使用此方法。结果，需要一个凝胶纯化步骤，该步骤可能会耗尽低抽象序列。为了避免对库的复杂性进行无意的更改，在连接步骤中阻止适配器二聚体形成是有利的。考虑到这一目标，我们提出了一种新型的NG RNA样品制备方法，该方法使用Trilink在核酸化学方面的专业知识来违反适配器 - 适配器结扎，同时允许有效地连接 图书馆的5'和3'末端的适配器问题。