researchandtesting.com/B2C2.html). Sequences less than 150 bp were removed for the BIBW2992 ic50 original bTEFAP method and less than 350 bp for the bTEFAP titanium method. To determine the identity of bacteria in the remaining VLU sequences, sequences were first queried using a distributed BLASTn .NET algorithm [32] against a database of high quality
16s bacterial sequences derived from NCBI. Database sequences were characterized as high quality based upon the criteria of RDP ver 9 [33]. Using a .NET and C# analysis pipeline the Ricolinostat purchase resulting BLASTn outputs were compiled, validated using taxonomic distance methods, and data reduction analysis performed
as described previously [9, 11, 13]. Rarefaction to estimate maximum diversity in wound using of 220 bp trimmed, non-ribosomal sequence depleted, chimera depleted, high quality reads was performed as described previously [8]. Bacterial identification Based upon the above BLASTn derived sequence identity (percent of total length query sequence which aligns with a given database sequence) and validated using taxonomic distance methods Selleck AZD1390 the bacteria were classified at the appropriate taxonomic levels based upon the following criteria. Sequences with identity scores, to known or well characterized 16S sequences, greater than 97% identity (<3% divergence) were resolved at the species level, between 95% and 97% at the genus level, between 90% and 95% at the family and between 80% and 90% at the order level. After resolving based upon these parameters, the percentage of each bacterial ID was individually analyzed for each wound providing relative abundance information within and among the VLU based upon relative numbers of reads within a given sample. Evaluations presented at a given taxonomic level, except species level, represent all sequences resolved to their primary genera
identification or their closest relative (where indicated). Metagenomics Metagenomic pyrosequencing reactions were performed at the Research and Testing Laboratory (Lubbock, Lumacaftor clinical trial TX). In short, DNA from a pool of 10 VLU preserved at -80°C, which had been previously analyzed using a 16s rDNA pyrosequencing microbial diversity approach [15] were further analyzed. DNA from these same 10 VLU samples were normalized and combined as described previously. Rather than perform bacterial 16s analysis as reported previously a metagenomic (or bulk sequencing) approach was performed using a half plate bulk sequencing reaction based upon FLX chemistry (Roche, Indianapolis, IN).