Everaert Celine, Verwilt Jasper, Verniers Kimberly, Vandamme Niels, Marcos Rubio Alvaro, Vandesompele Jo, Mestdagh Pieter
Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
Cancer Research Institute Ghent, Ghent University, Ghent, Belgium.
Biol Proced Online. 2023 Mar 8;25(1):7. doi: 10.1186/s12575-023-00193-3.
RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species.
We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3' end sequencing and long-read sequencing, and MALAT1 in single-cell 3' end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity.
Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.
RNA测序已成为转录组分析的金标准，但存在对低丰度转录本进行定量分析的固有挑战。与微阵列技术不同，RNA测序读数按转录本丰度成比例分配。因此，低丰度RNA会与高丰度（有时是无信息的）RNA种类竞争。
我们开发了一种基于高亲和力RNA结合寡核苷酸的简便策略，以阻断特定RNA转录本的逆转录和PCR扩增，从而在最终测序文库中大幅降低其丰度。为了证明我们方法的广泛应用潜力，我们将其应用于不同的转录本和文库制备策略，包括人血浆小RNA测序中的YRNA、3'端测序和长读长测序中的线粒体rRNA以及单细胞3'端测序中的MALAT1。我们证明该阻断策略高效、可重复、特异，并且通常能带来更好的转录组覆盖度和复杂度。
我们的方法除了在逆转录反应中简单添加阻断寡核苷酸外，无需对文库制备程序进行修改，因此可以轻松整合到几乎任何RNA测序文库制备方案中。
