Aoyagi Tomo, Hanada Satoshi, Itoh Hideomi, Sato Yuya, Ogata Atsushi, Friedrich Michael W, Kikuchi Yoshitomo, Hori Tomoyuki
Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Onogawa 16-1, Tsukuba, Ibaraki, 305-8569, Japan.
Environ Microbiol Rep. 2015 Apr;7(2):282-7. doi: 10.1111/1758-2229.12243. Epub 2015 Jan 23.
Stable isotope probing (SIP) of rRNA directly identifies microorganisms assimilating an isotopically labelled substrate. High-throughput DNA sequencing is available for label screening at high resolution and high sensitivity, yet its effectiveness and validity remain to be clarified. Here, we investigated whether the detection sensitivity of rRNA-SIP could be improved by using Illumina sequencing in place of terminal restriction fragment length polymorphism (T-RFLP) analysis. A dilution series of (13) C-labelled RNA from Escherichia coli (1-0.0001%) and unlabelled RNA from Bacillus subtilis was density separated and fractionated. Illumina sequencing of isopycnic centrifugation gradients was able to detect (13) C-labelled RNA in the heaviest fraction with a buoyant density of 1.798 g ml(-1) even at the mixing ratio of 0.001%, whereas the detection ability of T-RFLP was not lower than 0.5%. Quantitative reverse transcription polymerase chain reaction of the density-separated RNAs showed that (13) C-labelled RNAs at mixing ratios of 0.05-0.001% had definitely accumulated in the heaviest fraction. Consequently, high-throughput sequencing provided up to 500-fold higher sensitivity for screening of (13) C-labelled RNA than T-RFLP. Ultra-high-sensitivity rRNA-SIP represents a clear advance towards a more complete understanding of microbial ecosystem function, including the ecophysiology of rare microorganisms in various natural environments.
rRNA的稳定同位素探测（SIP）可直接鉴定同化同位素标记底物的微生物。高通量DNA测序可用于高分辨率和高灵敏度的标记筛选，但其有效性和准确性仍有待明确。在此，我们研究了使用Illumina测序代替末端限制性片段长度多态性（T-RFLP）分析是否能提高rRNA-SIP的检测灵敏度。对来自大肠杆菌的一系列稀释的（13）C标记RNA（1%-0.0001%）和来自枯草芽孢杆菌的未标记RNA进行密度分离和分级。等密度离心梯度的Illumina测序能够在浮力密度为1.798 g/ml的最重级分中检测到（13）C标记的RNA，即使在混合比例为0.001%时也是如此，而T-RFLP的检测能力不低于0.5%。对密度分离的RNA进行定量逆转录聚合酶链反应表明，混合比例为0.05%-0.001%的（13）C标记RNA肯定在最重级分中积累。因此，高通量测序在筛选（13）C标记RNA方面的灵敏度比T-RFLP高500倍。超高灵敏度rRNA-SIP代表了在更全面理解微生物生态系统功能方面的明显进展，包括各种自然环境中稀有微生物的生态生理学。
