摘要
Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high rRNA:mRNA ratio (80–90%: ~10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA, and therefore, requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here, we show that after the discontinuation of the previous version of RiboZero (Illumina, San Diego, CA, USA) that was useful in partially or completely depleting rRNA from archaea, archaeal transcriptomics studies have experienced a slowdown. To overcome this limitation, here, we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper, we provide insights into the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.
摘要译文
尽管最近对古细菌进行了激烈的研究兴趣，但科学界在研究基因组规模的基因表达实验的研究中经历了RNA-Seq的瓶颈，这是由于缺乏商业和专门设计的RRNA耗竭试剂盒。高rRNA：原核生物中的mRNA比率（80–90％：〜10％）阻碍了全局转录组分析。核本序列不足会导致mRNA的低序列覆盖范围，因此，需要更高数量的复制样品和/或测序读取，以达到有关病例和对照样品之间差异基因表达的重要性的统计上可靠的结论。在这里，我们表明，在先前版本的Ribozero（Illumina，Illumina，San Diego，CA，美国）中止后，对古细菌的部分或完全耗尽了RRNA，古细菌转录组学研究经历了放缓。为了克服这一局限性，我们在这里分析了来自三个不同商业供应商的四个不同杂交试剂盒的效率，每个供应商都有两组序列特异性探针，以从四种不同种类的卤素古细菌中去除rRNA。我们得出的结论是，使用当前可用工具的转录组成功的关键是rRNA序列杂交的探针特异性。在本文的情况下，我们提供了对古细菌社区的见解，以根据感兴趣的古细菌物种选择某些试剂和策略。这些方法可提高RNA-seq敏感性，并增强对低丰度转录本的检测。
Mar Martinez Pastor;Saaz Sakrikar;Deyra N. Rodriguez;Amy K. Schmid. Comparative Analysis of rRNA Removal Methods for RNA-Seq Differential Expression in Halophilic Archaea[J]. Biomolecules, 2022,12(5): 682