Computational protocol: Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota

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Protocol publication

[…] Experimental sequences were processed using a custom PERL script, which performed the following: the script filtered the sequence files and retained only sequences that were 200-nt or longer. It then searched for a single barcode sequence in each FASTA sequence, binned each sequence accordingly, and scanned each binned sequence for the 16S forward primer sequence. The script then trimmed off the forward primer sequence and oriented the remaining sequence such that all sequences begin with the 5′ end according to standard sense strand conventions. As a result of our processing, sequences that were shorter than 200-nt or had multiple barcode or primer motifs were excluded from the analysis. We included only sequences with the forward primer motif to ensure that the highly informative V3 region was available for taxonomic assignment. The trimmed sequences from each barcode bin were aligned using the NAST alignment tool (http://greengenes.lbl.gov) . After alignment, the number of sequences examined per wound sample was equilibrized to 300 sequences by sampling randomly without replacement to facilitate subsequent taxa abundance analyses. Samples with fewer than 300 sequences were excluded. The cutoff of n = 300 was established based on richness (rarefaction) and diversity (Shannon-Weaver Index) analyses using DOTUR , which indicated that samples were sufficiently sampled after ≥300 sequences. [...] Unaligned, sequences in the equilibrated dataset were given taxonomic assignments at a bootstrap confidence range of ≥95% using the Ribosomal Database Project's Naïve Bayesian Classifier tool (RDP classifier) , . [...] Distance matrices based on taxa abundance were generated with the Dnadist tool of PHYLIP 3.67 using the default settings . Rarefaction and Shannon Weaver index estimations were determined by DOTUR and plotted in Microsoft Excel (Microsoft Corp., USA). […]

Pipeline specifications

Software tools DOTUR, RDP Classifier, PHYLIP
Application 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Diabetes Mellitus, Neural Tube Defects