Mäki Anita, Tiirola Marja
Department of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, PO Box 35, FI-40014, Finland.
Biotechniques. 2018 Oct;65(4):219-223. doi: 10.2144/btn-2018-0082.
Ribosomal RNA analysis is a useful tool for characterization of microbial communities. However, the lack of broad-range primers has hampered the simultaneous analysis of eukaryotic and prokaryotic members by amplicon sequencing. We present a complete workflow for directional, primer-independent sequencing of size-selected small subunit ribosomal RNA fragments. The library preparation protocol includes gel extraction of the target RNA, ligation of an RNA oligo to the 5'-end of the target, and cDNA synthesis with a tailed random-hexamer primer and further barcoding. The sequencing results of a phytoplankton mock community showed a highly similar profile to the biomass indicators. This method has universal potential for microbiome studies, and is compatible for the 5'-end sequencing of other RNA types with minimum library preparation costs.
核糖体RNA分析是表征微生物群落的一种有用工具。然而，缺乏广泛适用的引物阻碍了通过扩增子测序对真核生物和原核生物成员进行同步分析。我们提出了一种完整的工作流程，用于对大小选择的小亚基核糖体RNA片段进行定向、无需引物的测序。文库制备方案包括对目标RNA进行凝胶提取、将RNA寡核苷酸连接到目标的5'端，以及使用带尾随机六聚体引物进行cDNA合成并进一步添加条形码。浮游植物模拟群落的测序结果显示出与生物量指标高度相似的图谱。该方法在微生物组研究中具有广泛的应用潜力，并且以最低的文库制备成本适用于其他RNA类型的5'端测序。
