Use of pyrosequencing and DNA barcodes to monitor variations in Firmicutes and Bacteroidetes communities in the gut microbiota of obese humans.
BACKGROUND
Recent studies of 16S rRNA genes in the mammalian gut microbiota distinguished a higher Firmicutes/Bacteroidetes ratio in obese individuals compared to lean individuals. This ratio was estimated using a clonal Sanger sequencing approach which is time-consuming and requires laborious data analysis. In contrast, new high-throughput pyrosequencing technology offers an inexpensive alternative to clonal Sanger sequencing and would significantly advance our understanding of obesity via the development of a clinical diagnostic method. Here we present a cost-effective method that combines 16S rRNA pyrosequencing and DNA barcodes of the Firmicutes and Bacteroidetes 16S rRNA genes to determine the Firmicutes/Bacteroidetes ratio in the gut microbiota of obese humans.


RESULTS
The main result was the identification of DNA barcodes targeting the Firmicutes and Bacteroidetes phyla. These barcodes were validated using previously published 16S rRNA gut microbiota clone libraries. In addition, an accurate F/B ratio was found when the DNA barcodes were applied to short pyrosequencing reads of published gut metagenomes. Finally, the barcodes were utilized to define the F/B ratio of 16S rRNA pyrosequencing data generated from brain abscess pus and cystic fibrosis sputum.


CONCLUSIONS
Using DNA barcodes of Bacteroidetes and Firmicutes 16S rRNA genes combined with pyrosequencing is a cost-effective method for monitoring relevant changes in the relative abundance of Firmicutes and Bacteroidetes bacterial communities in microbial ecosystems.
Armougom
Fabrice
F
URMITE - UMR CNRS 6236, IRD 3R198, Université de la Méditerranée, Faculté de médecine, Marseille, France. fabrice.armougom@univmed.fr
Raoult
Didier
D
D015373:Bacterial Typing Techniques / Q000379:methods; D041963:Bacteroidetes / Q000145:classification / Q000235:genetics*; D041981:Gastrointestinal Tract / Q000382:microbiology*; D020459:Genes, rRNA; D005819:Genetic Markers; D014644:Genetic Variation; D006801:Humans; D009765:Obesity / Q000382:microbiology*; D012329:RNA, Bacterial / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
D005819:Genetic Markers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S