Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota.
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.
Matsumoto
Mitsuharu
M
Fundamental Research Laboratory, Kyodo Milk Industry Co. Ltd., Hinode, Tokyo 190-0182, Japan. m-matumoto@meito.co.jp
Sakamoto
Mitsuo
M
Hayashi
Hidenori
H
Benno
Yoshimi
Y
D000328:Adult; D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D003106:Colon / Q000382:microbiology*; D004269:DNA, Bacterial / Q000235:genetics; D030541:Databases, Genetic; D015723:Gene Library; D005798:Genes, Bacterial; D006801:Humans; D008297:Male; D010802:Phylogeny; D012150:Polymorphism, Restriction Fragment Length; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S