Department of Pediatric Pneumology, Allergology, and Neonatology, Hannover Medical School, Hannover, Germany.
Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research, Hannover Medical School, Hannover, Germany.
J Bacteriol. 2022 Jan 18;204(1):e0041821. doi: 10.1128/JB.00418-21. Epub 2021 Nov 15.
The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNA, and the tRNA gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs. Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that-if replicated in other species-will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria.
采用纳米孔平台直接对 RNA 进行测序，对浮游生长中期和早期静止期铜绿假单胞菌克隆 C 株 NN2 和 SG17M 的转录组进行了评估，并与 Illumina 平台上已建立的短读 cDNA 测序进行了比较。结果发现，有 50%到 90%的有意义 RNA 实际上是 rRNA 分子，其次是相似比例的 mRNA 转录本和非编码 RNA。这两个平台检测到未带电 tRNA 和 29 个尚未描述的反义 tRNA 的比例相似。例如，最稀有的精氨酸密码子与最丰富的 tRNA 配对，而最常见的精氨酸密码子则缺少 tRNA 基因。超过 90%的反义 RNA 分子与编码序列互补。反义 RNA 均匀分布在基因组中。直接 RNA 测序在指数和静止生长过程中鉴定了超过 4000 个独特的不重叠反义 RNA。除了小于 200 个碱基的高度表达小反义 RNA 外，还测序了长反义 RNA 群体，其覆盖范围很广（几百到几千个碱基），并且可以与几个基因的连续序列互补。总之，直接 RNA 测序鉴定了尚未描述的 RNA 分子以及 tRNA 和有意义及反义 RNA 库的意外组成。 细菌的全基因组基因表达通常通过对逆转录 RNA 制备的大小选择 cDNA 片段文库进行高通量测序来研究。然而，rRNA 的耗尽、酶促逆转录以及文库制备过程中的片段化、大小选择和扩增，导致 RNA 池初始组成信息不可避免地丢失。我们证明，Nanopore 平台上的直接 RNA 测序可以克服这些限制。对总 RNA 的 Nanopore 测序为铜绿假单胞菌转录组提供了新的见解，如果在其他物种中复制，将改变我们对细菌 RNA 世界的看法。反义转移信使 RNA (tmRNA)、tRNA 和 mRNA 的 sense-antisense 对的发现表明细菌中存在进一步的未知水平的基因调控。
