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Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota.

Mitsuharu Matsumoto·Mitsuo Sakamoto·Hidenori Hayashi·Yoshimi Benno
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Abstract

Various molecular-biological approaches using the 16S rRNA gene sequence have been used for the analysis of human colonic microbiota. Terminal- restriction fragment length polymorphism (T-RFLP) analysis is suitable for a rapid comparison of complex bacterial communities. Terminal-restriction fragment (T-RF) length can be calculated from a known sequence, thus one can predict bacterial species on the basis of their T-RF length by this analysis. The aim of this study was to build a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota (PAD-HCM), and to demonstrate the effectiveness of PAD-HCM compared with the results of 16S rRNA gene clone library analysis. PAD-HCM was completed to include 342 sequence data obtained using four restriction enzymes. Approximately 80% of the total clones detected by 16S rRNA gene clone library analysis were the same bacterial species or phylotypes as those assigned from T-RF using PAD-HCM. Moreover, large T-RFs consisted of common species or phylotypes detected by both analytical methods. All pseudo-T-RFs identified by mung bean nuclease digestion could not be assigned to a bacterial species or phylotype, and this finding shows that pseudo-T-RFs can also be predicted using PAD-HCM. We conclude that PAD-HCM built in this study enables the prediction of T-RFs at the species level including difficult-to-culture bacteria, and that it is very useful for the T-RFLP analysis of human colonic microbiota.

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AdultBacteriaBase SequenceColonDNA, BacterialDatabases, GeneticGene LibraryGenes, BacterialHumansMalePhylogenyPolymorphism, Restriction Fragment LengthRNA, BacterialRNA, Ribosomal, 16SJournal Article

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BibTeXRIS

Mitsuharu Matsumoto, Mitsuo Sakamoto, Hidenori Hayashi, Yoshimi Benno. 2005-01-11. Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota.. https://doi.org/10.1016/j.mimet.2004.12.009

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