Ultra-high-sensitivity stable-isotope probing of rRNA by high-throughput sequencing of isopycnic centrifugation gradients.

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.