Department of Environmental Science, Policy, and Management, University of California, Berkeley, Berkeley, California, United States of America.
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States of America.
PLoS One. 2020 Mar 4;15(3):e0229537. doi: 10.1371/journal.pone.0229537. eCollection 2020.
Microbes alter their transcriptomic profiles in response to the environment. The physiological conditions experienced by a microbial community can thus be inferred using meta-transcriptomic sequencing by comparing transcription levels of specifically chosen genes. However, this analysis requires accurate reference genomes to identify the specific genes from which RNA reads originate. In addition, such an analysis should avoid biases in transcript counts related to differences in organism abundance. In this study we describe an approach to address these difficulties. Sample-specific meta-genomic assembled genomes (MAGs) were used as reference genomes to accurately identify the origin of RNA reads, and transcript ratios of genes with opposite transcription responses were compared to eliminate biases related to differences in organismal abundance, an approach hereafter named the "diametric ratio" method. We used this approach to probe the environmental conditions experienced by Escherichia spp. in the gut of 4 premature infants, 2 of whom developed necrotizing enterocolitis (NEC), a severe inflammatory intestinal disease. We analyzed twenty fecal samples taken from four premature infants (4-6 time points from each infant), and found significantly higher diametric ratios of genes associated with low oxygen levels in samples of infants later diagnosed with NEC than in samples without NEC. We also show this method can be used for examining other physiological conditions, such as exposure to nitric oxide and osmotic pressure. These study results should be treated with caution, due to the presence of confounding factors that might also distinguish between NEC and control infants. Nevertheless, together with benchmarking analyses, we show here that the diametric ratio approach can be applied for evaluating the physiological conditions experienced by microbes in situ. Results from similar studies can be further applied for designing diagnostic methods to detect NEC in its early developmental stages.
微生物会根据环境改变其转录组谱。因此，通过比较特定选择基因的转录水平，可利用元转录组测序来推断微生物群落经历的生理条件。然而，这种分析需要准确的参考基因组来识别 RNA 读取源自特定基因。此外，这种分析应避免与生物丰度差异相关的转录计数偏倚。在这项研究中，我们描述了一种解决这些困难的方法。使用特定于样本的宏基因组组装基因组 (MAG) 作为参考基因组，以准确识别 RNA 读取的来源，并且比较具有相反转录响应的基因的转录比值，以消除与生物丰度差异相关的偏倚，这种方法此后被称为“直径比”方法。我们使用这种方法来探测在 4 名早产儿肠道中埃希氏菌属经历的环境条件，其中 2 名早产儿患坏死性小肠结肠炎（NEC），一种严重的炎症性肠道疾病。我们分析了来自 4 名早产儿的 20 个粪便样本（每个婴儿 4-6 个时间点），发现与低氧水平相关的基因的直径比在后来被诊断为 NEC 的婴儿的样本中明显高于没有 NEC 的样本。我们还表明，该方法可用于检查其他生理条件，例如暴露于一氧化氮和渗透压。由于可能区分 NEC 婴儿和对照婴儿的混杂因素的存在，这些研究结果应谨慎对待。尽管如此，我们还结合基准分析表明，直径比方法可用于评估原位微生物经历的生理条件。类似研究的结果可以进一步应用于设计在早期发育阶段检测 NEC 的诊断方法。
