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Ultra‐high‐sensitivity stable‐isotope probing of rRNA by high‐throughput sequencing of isopycnic centrifugation gradients
Author(s) -
Aoyagi Tomo,
Hanada Satoshi,
Itoh Hideomi,
Sato Yuya,
Ogata Atsushi,
Friedrich Michael W.,
Kikuchi Yoshitomo,
Hori Tomoyuki
Publication year - 2015
Publication title -
environmental microbiology reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.229
H-Index - 69
ISSN - 1758-2229
DOI - 10.1111/1758-2229.12243
Subject(s) - biology , ribosomal rna , isopycnic , illumina dye sequencing , rna , differential centrifugation , stable isotope probing , dna sequencing , terminal restriction fragment length polymorphism , deep sequencing , microbiology and biotechnology , centrifugation , chromatography , restriction fragment length polymorphism , polymerase chain reaction , dna , genetics , microorganism , gene , chemistry , biochemistry , genome , bacteria
Summary 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 E scherichia coli (1–0.0001%) and unlabelled RNA from B acillus 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 RNA s 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.