Pyrosequence-Based 16S rRNA Profiling of Gastro-Intestinal Microbiota

Abstract

Pyrosequence-based 16S rRNA profiling has become a very powerful tool for visualizing the community structure of gastro-intestinal (GI) tract microbiota. The system was established with newly designed universal primers with barcode sequences and newly modified algorithms to convert batch sequence data to bacterial population data. In silico primer match simulation indicates that the primers, Q-968F, Q-1046R, and Q-1390R, match to almost of all 16S rRNAs in the database within one base mismatch, with especially high coverage ratios for the four biggest common phyla in human GI tract. Also, the new SeqmatchQ100 algorithm correctly assigns almost all of the target 60-base sequences of the 16S rRNA V6 region to the corresponding genus except for those from the Enterobacteriaceae family and the Enterococcus genus. Furthermore, the SeqmatchQ400 algorithm efficiently provides species-level population data from a 400-base sequence of the 16S rRNA V6―V8 region with the exceptions of the Enterobacteriaceae and Enterococcaceae families. A barcode-sequence tag strategy was used to analyze up to 128 samples at a time. With these newly prepared tools, pyrosequence-based 16S rRNA profiling displays community structures of GI-tract microbiota. For instance, establishment of bifidus flora in newborn infants and dynamics in the microbial community structure after weaning were effectively demonstrated by 16S rRNA profiling. In future, this analytical system should be of use for monitoring changes in GI-tract bacterial composition which may be influenced by diets, drugs, or sickness.