A high amount of actinobacterial sequences recovered If the proportional amount of DNA in

each fraction is taken into account in estimating the abundance of phyla, 28.5% of the sequences would affiliate with Actinobacteria. Since the %G+C profile fractions represent individual cloning and sequencing experiments, in which an equal amount of clones were sequenced despite the different proportional amounts of DNA within the fractions, quantitative conclusions should be drawn carefully. However, %G+C fractions 50–70 were dominated by Actinobacteria, comprising 41% of the total DNA in the original sample fractioned (Figures 1 and 2, Additional file 1). The %G+C fractions 30–50 yield a similar phylotype TSA HDAC manufacturer distribution as the unfractioned library (Figure find more 2). These fractions, accounting

for 54% of the profiled DNA, are dominated by the Firmicutes (Clostridium clusters XIV and IV) (Figure 1 and 2). The relatively high proportion of actinobacterial sequences (26.6%) and phylotypes (65) identified in the combined sequence data of the %G+C fractioned sample exceed all previous estimations. In a metagenomic study by Gill and colleagues [14], 20.5% of 132 16S rRNA sequences from random shotgun assemblies affiliated with 10 phylotypes of Actinobacteria whereas no Bacteroidetes was detected. In accordance with our results, also a pyrosequencing study by Andersson and colleagues [16], the Actinobacteria (14.6%), dominated by a few phylotypes, outnumbered Bacteroidetes (2.5%). By contrast, in most of the earlier published studies

on human faecal samples applying 16S rRNA gene amplification, cloning and sequencing, the relative amount of Actinobacteria has been 0–6% of the detected intestinal microbiota [12, 25–33]. Thus, the proportion of sequences affiliating with Actinobacteria (3.5%) in the unfractioned sample analysed in this study is A-1210477 comparable with previous estimations applying conventional 16S rRNA cloning and sequencing without %G+C fractioning. Order Coriobacteriales abundant within Actinobacteria We observed that several clones in the high %G+C fractions (60–70% G+C content) were next tricky to sequence due to extremely G+C rich regions. These clones turned out to be members of order Coriobacteriales, which have been rare or absent in earlier 16S rRNA gene -based clone libraries of the intestinal microbiota. Over half of the actinobacterial OTUs in our study belonged to the order Coriobacteriales. Harmsen et al. [34] earlier suggested that applications based on 16S rRNA gene cloning as well as other methods of molecular biology may overlook the presence of the family Coriobacteriaceae in the human GI tract and they designed a group-specific probe for Atopobium (Ato291), covering most of the Coriobacteriaceae, the Coriobacterium group.