Organism-specific depletion of highly abundant RNA species from bacterial total RNA. Organism-specific depletion of highly abundant RNA species from bacterial total RNA

We assess the mRNA-enrichment performance of a custom-made non-mRNA depletion protocol in comparison to a commercially available mRNA-enrichment kit (Ribo-off, Vazyme). Whereas most available kits focus only on removal of rRNA, our method also targets the transfer-messenger RNA (tmRNA). tmRNA was shown to consume up to 25% of the reads in RNA-sequencing data of Pseudomonas aeruginosa. Our established depletion technique is based on the targeting of overly abundant RNA species (16S and23S rRNA, tmRNA) in total RNA preparations of Pseudomonas aeruginosa PA14 by hybridization with organism-specific DNA probes and subsequent degradation by RNase H treatment. While introducing no systematic bias into the gene expression profile we were able to increase the mRNA read share of the total reads in the samples treated with our mRNA-enrichment protocol to 93% - 99%. Therefore, our custom-made depletion technique outcompeted the commercially available reference kit (72% mRNA share) and represents a cost-efficient mRNA-enrichment method for high-throughput next-generation sequencing. Overall design: Total RNA preparations from Pseudomonas aeruginosa PA14 harvested in exponential or stationary growth phase were treated with a custom-made mRNA enrichment protocol or a reference depletion kit (Ribo-off, Vazyme). Experiment was performed in two biological and two technical replicates.

本研究评估了定制化非mRNA去除方案的mRNA富集效果，并与商业化mRNA富集试剂盒（Ribo-off, Vazyme）进行对比。多数现有试剂盒仅针对核糖体RNA（ribosomal RNA, rRNA）进行去除，而本方法同时靶向转运信使RNA（transfer-messenger RNA, tmRNA）。已有研究表明，铜绿假单胞菌（Pseudomonas aeruginosa）的RNA测序数据中，tmRNA可占据最高达25%的测序读段。我们开发的去除技术以铜绿假单胞菌PA14总RNA样品中丰度过高的RNA物种（16S、23S rRNA及tmRNA）为靶点，通过与物种特异性DNA探针杂交，再经核糖核酸酶H（RNase H）处理实现降解。该方法不会对基因表达谱引入系统性偏差，经定制化mRNA富集方案处理后的样品，其总读段中的mRNA占比可提升至93%~99%。因此，本定制化去除方案的性能优于商业化对照试剂盒（其mRNA占比仅为72%），可作为一种适用于高通量下一代测序的经济高效mRNA富集方法。实验设计：分别收集处于指数生长期与稳定生长期的铜绿假单胞菌PA14的总RNA样品，经定制化mRNA富集方案或对照去除试剂盒（Ribo-off, Vazyme）处理。本实验设置2次生物学重复与2次技术重复。