The results of the qPCR were provided to us in the form of relative ratios of each detected bacterium in the sample and these results compared Rabusertib nmr to the corresponding bTEFAP bacterial ratio data. In short the percentages of the key bacteria detected using bTEFAP analysis were correlated (0.78, P = 0.001) with the relative percentages determined using qPCR. This provides an indication of the validity of the bTEFAP data. Metagenomics We evaluated, using a bulk pyrosequencing metagenomics approach, a uniformly compiled pool of 10 VLU DNA extractions. A total
of 178,610 individual reads were generated averaging 248 bp. There were 42,441 reads that could be assigned taxonomic designations. Of those reads assigned to a taxonomic designation the majority (30,141) fell into the chordata, which represents human genetic information confirmed based upon subsequence BLASTn and BLASTx designations to homo sapiens genomic data contained within NCBI. The remaining reads were utilized to generate an evaluation of the microbial population within these 10 VLU samples. There were 7,497 reads, which were assigned to bacteria, which was evaluated at the class level for the subsequent comparisons. Table 1 provides a comparative breakdown at the bacterial class level of bTEFAP analyses and the metagenomic analysis. There was good overall relationship (r-squared = 0.74) with what was predicted in the 10-sample VLU pool using metagenomic data and what was detected using
the same 10 sample pool analyzed in our previous work using bTEFAP . Interestingly, there was also Y27632 a positive relationship
at the same class taxonomic level between the 10-sample pool and the averages of the 40 VLU samples at the class level (Table 2). Table 2 The 10 sample pool metagenomic analysis comparison to bTEFAP 10 sample pool and bTEFAP 40 sample averages at the taxonomic class level. Class bTEFAP 10 pool % Metagenomics 10 pool % bTEFAP 40 avg. % Bacilli 4.5 4.6 29 Gammaproteobacteria 54 37.4 25 Clostridia 1.1 4.4 12 Betaproteobacteria 2.6 3.6 0.1 Actinobacteria (class) 1.1 19.1 12 Alphaproteobacteria 1.4 7.6 05 deltaproteobacteria 5.4 7.5 0.14 selleck kinase inhibitor Epsilonproteobacteria 2 13 0.24 Bacteroidetes 10.5 6.1 17.9 other 17.2 8.6 3.5 This stiripentol table shows the difference in metagenomic and 16s pyrosequencing approach described previously . Also shown is the averages related to the 40 individual samples for comparison. The R-squared = 0.74 for correlation between bTEFAP and metagenomics at the class level in the 10 pooled samples. Further analysis of the metagenomic data in relation to other microorganisms provided additional interesting information. A relatively high number of genes (2566) mapped to Apicomplexa (most closely related to Plasmodium yoelii) were detected. Fungi (most closely related to 3 yeast including Candida albicans, Candida glabrata and Aspergillus spp with some reads showing very distant relationships to Yarrowia spp and Magnaporthe spp) made up 668 reads.