A method for preparing unbiased miRNA sequencing libraries

描述（由申请人提供）：该提案解决了通过下一代测序（NGS）进行的microRNA（miRNA）和其他小RNA表达谱分析中的偏倚问题。由于miRNA表达的失调与癌症和某些其他疾病有关，因此所有miRNA序列的准确表达谱对于理解miRNA生物学和开发新的生物标志物和治疗靶点非常重要。NGS是目前最全面的数字基因表达谱分析方法，用于发现新的miRNA序列，鉴定序列变体和定量已知的miRNA。与其他表达谱分析平台（如微阵列或RT-qPCR）不同，NGS结合了无限多路复用能力、单分子灵敏度、上级动态范围和真正的序列特异性，而无需预先了解miRNA序列。然而，NGS表达谱数据低估了样品中许多miRNA的量多达10，000倍。了解样本中的绝对丰度，而不仅仅是样本之间的相对变化，对于可靠地鉴定作为生物标志物或药物靶点候选物的miRNA是重要的。在NGS中，衔接子与RNA末端的酶促连接是miRNA定量中发生大部分偏倚的步骤。导致这种连接偏倚的主要因素是miRNA的分子内折叠和miRNA与接头之间的分子间折叠，其影响连接酶接近和连接miRNA末端的能力。因此，需要新的、更准确的方法，并且应该重新评估大多数先前的小RNA谱分析实验。为了解决这些问题，我们提出了一种新的方法，miR-ACS（miRNA-Adapter Circularization and Sequencing），用于制备miRNA测序文库。miR-ACS的关键特征包括（i）仅用单个衔接子连接miRNAs;（ii）环化miRNAs;（iii）连接miRNAs。 （iii）消除未连接至miRNA-接头连接产物的接头种类; （iv）环状miRNA衔接子加合物的RT-PCR扩增，随后凝胶纯化适于NGS测序的扩增子。MiR-ACS有可能基本消除miRNA测序偏倚，并显著减少不相关miRNA测序读数的数量（允许平行测序更多样本）。这些特征还可以降低成本并增加NGS测序的通量。尽管miR-ACS适用于任何样本中miRNA或其他小RNA的表达谱分析，但为了验证概念，我们专注于与前列腺癌相关的样本。

DESCRIPTION (provided by applicant): This proposal addresses the problem of bias in the expression profiling of microRNAs (miRNAs) and other small RNAs by next-generation sequencing (NGS). Because dysregulation of miRNA expression has been implicated in cancer and certain 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 digital gene expression profiling, and is used for the discovery of novel miRNA sequences, identification of sequence variants, and the quantification of known miRNAs. Unlike other expression profiling platforms such as microarrays or RT-qPCR, NGS combines unlimited multiplexing capability, single-molecule sensitivity, a superior dynamic range, and true sequence specificity without requiring prior knowledge of miRNA sequences. However, NGS expression profiling data underestimate the amount of many miRNAs in a sample by as much as 10,000-fold. Knowledge of the absolute abundances in samples, and not just the relative changes between samples, is important for reliable identification of miRNAs as biomarkers or drug-target candidates. In NGS, the enzymatic ligation of adapters to the RNA ends is the step where most of the bias in miRNA quantification occurs. The major factors contributing to this ligation bias are intramolecular folding of the miRNAs and intermolecular folding between miRNAs and the adapters, which affect the ability of ligation enzymes 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 propose a new approach, miR-ACS (miRNA-Adapter Circularization and Sequencing), for preparing miRNA sequencing libraries. Key features of miR-ACS include (i) ligation of miRNAs with only a single adapter; (ii) circularization of the miRNA-adapter ligation product; (iii) elimination of adapter species that are not ligated to miRNAs; and (iv) RT-PCR amplification of the circular miRNA adapter adduct followed by gel-purification of amplicons suitable for NGS sequencing. MiR-ACS has the potential to essentially eliminate miRNA sequencing bias and significantly reduce the number of irrelevant miRNAs sequencing reads (allowing the sequencing of more samples in parallel). These features may also reduce cost and increase the throughput of NGS sequencing. Although miR-ACS is applicable to expression profiling of miRNA or other small RNAs in any sample, for proof-of-concept we focus on samples associated with prostate cancer.