A method for preparing unbiased miRNA sequencing libraries

描述（由申请人提供）：本提案旨在通过下一代测序（NGS）解决microRNAs （miRNAs）和其他小rna表达谱中的偏差问题。由于miRNA表达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生物学以及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方法，用于发现新的miRNA序列、鉴定序列变异和定量已知miRNA。与其他表达谱分析平台（如微阵列或RT-qPCR）不同，NGS结合了无限的多路复用能力、单分子灵敏度、优越的动态范围和真正的序列特异性，而无需事先了解miRNA序列。然而，NGS表达谱数据低估了样品中许多mirna的数量多达10,000倍。了解样品中的绝对丰度，而不仅仅是样品之间的相对变化，对于可靠地鉴定mirna作为生物标志物或候选药物靶标非常重要。在NGS中，转接器与RNA末端的酶联是miRNA定量中大多数偏差发生的步骤。导致这种连接偏差的主要因素是miRNA的分子内折叠和miRNA与适配器之间的分子间折叠，这影响了连接酶接近和连接miRNA末端的能力。因此，需要新的、更精确的方法，并且大多数以前的小RNA分析实验应该重新评估。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的主要特征包括(i)仅通过一个适配器连接mirna；(2)环化

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.