A method for preparing unbiased miRNA sequencing libraries (miR-ACS)

摘要 这一建议解决了小RNA下一代测序(NGS)中的序列偏差问题 例如microRNAs(MiRNAs)以及较大RNAs的片段。因为miRNA的失调 表达与癌症和其他疾病有关，所有miRNA的准确表达谱 序列对于理解miRNA生物学和开发新的生物标记物和 治疗靶点。NGS是目前发现和表达谱分析最全面的方法 小的RNA序列。然而，NGS表达谱数据低估了大多数 一个样本中的miRNAs，有些增加了10,000倍。对样品中真实丰度的了解，而不是 仅仅是样本之间的相对变化，对于可靠地将miRNAs识别为生物标志物或 毒品目标候选人。无偏检测的其他优势包括能够发现新的RNA和 检测当前NGS方法无法检测到的低丰度RNA，特别是在具有 低浓度的RNA。目前可用的制备RNA测序方法的偏倚来源 NGS文库是两个测序适配子与样本的低效和依赖于序列的连接 RNA。造成这种连接偏差的主要因素是miRNAs的分子内折叠和 MiRNAs和接头之间的分子间折叠，影响连接酶进入和连接的能力 MiRNA结束了。因此，需要新的、更准确的方法，以及大多数以前的小RNA图谱 实验应该重新评估。为了解决这些问题，我们正在开发一种新的方法，miR- ACS(miRNA-Adapter环化和测序)，用于制备无偏序文库，即 适用于miRNA和其他小RNA以及一般RNA中使用的大RNA的小片段- 序列号。MiR-ACS的主要特征包括：(I)仅使用单个组合适配器(CAD)连接miRNAs 组合用于Illumina测序的标准3‘和5’适配子的序列，产生miRNA-CAD 连接产物；(Ii)miRNA-CAD产物的环化；(Iii)阻断非 连接到miRNAs；和(Iv)环状miRNA-CAD产物的RT-PCR扩增，以产生标准 含有单个RNA特异性序列插入的测序扩增，侧翼为5‘-和3’-接头 序列。在第一阶段，我们通过以下方式论证了miR-ACS方法(概念验证)的可行性 与目前最好的文库制备方法相比，大大降低了miRNA测序偏差。同相 II我们将彻底优化miR-ACS，以最大限度地减少偏差，并允许对更多品种进行测序 具有非常低的RNA输入的RNA(大小高达150个核苷酸)。此外，我们会简化议定书，以方便 它被用户采用，并具有商业可行性。

ABSTRACT This proposal addresses the problem of sequence bias in next-generation sequencing (NGS) of small RNAs such as microRNAs (miRNAs) as well as fragments of larger RNAs. Because dysregulation of miRNA expression has been implicated in cancer and other diseases, accurate expression profiling of all miRNA sequences is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. NGS is currently the most comprehensive approach for discovery and expression profiling of small RNA sequences. However, NGS expression profiling data underestimate the abundance of most miRNAs in a sample, some by as much as 10,000-fold. Knowledge of the true abundances in samples, and not just the relative changes between samples, is important for reliable identification of miRNAs as biomarkers or drug-target candidates. Other advantages of unbiased detection include the ability to discover novel RNAs and detect low-abundance RNAs that cannot be detected by current NGS methods, especially in samples with a low concentration of RNA. The source of bias in currently available methods of preparation RNA sequencing libraries for NGS is inefficient and sequence-dependent ligation of the two sequencing adapters to the sample RNAs. The major factors contributing to this ligation bias are intramolecular folding of the miRNAs and intermolecular folding between miRNAs and adapters, which affect the ability of the ligase to access and ligate the miRNA ends. Thus there is a need for new, more accurate methods, and most previous small RNA profiling experiments should be re-evaluated. To address these problems, we are developing a new approach, miR- ACS (miRNA-Adapter Circularization and Sequencing), for preparing unbiased sequencing libraries that is applicable to miRNAs and other small RNAs as well as small fragments of large RNAs used in general RNA- Seq. Key features of miR-ACS include (i) ligation of miRNAs with only a single combo adapter (CAD) that combines sequences of the standard 3'- and 5'-adapters used for Illumina sequencing, producing miRNA-CAD ligation products; (ii) circularization of the miRNA-CAD products; (iii) blocking of free CAD species that are not ligated to miRNAs; and (iv) RT-PCR amplification of the circular miRNA-CAD products to produce standard sequencing amplicons containing a single RNA-specific sequence insert flanked by the 5'- and 3'-adapter sequences. In Phase I, we have demonstrated the feasibility of the miR-ACS approach (proof-of-concept) by greatly reducing the miRNA sequencing bias in comparison to the best current library prep methods. In Phase II we will thoroughly optimize miR-ACS to maximize bias reduction and to allow sequencing of a larger variety of RNAs (up to 150 nt in size) with very low RNA inputs. In addition, we will streamline the protocol to facilitate its adoption by users and for commercial viability.