Computational protocol: Microbiological pattern of arterial catheters in the intensive care unit

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Protocol publication

[…] The 16S rRNA gene clones of the arterial catheters were divided into two groups, i.e., uncolonised ACs and colonised ACs. The 16S rRNA gene sequences obtained were manually proofread, corrected and edited to start and end with the corresponding primer nucleotide (using reverse complement transform if necessary) using BioEdit []. Sequences with incorrect inserts or with ambiguous bases were excluded from further sequence analyses. Vector sequences detected by cross match were trimmed off. Trimmed, assembled sequences were then aligned to a core set of sequences using the NAST alignment tool on the Greengens website (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi). All 16S rRNA gene sequences were screened for potential chimeras using BELLEROPHON which was also available on the Greengens website [] and sequences flagged as potential chimeras were discarded from further analysis. Sequences were compared to the NCBI GenBank database using the BLAST program. All examined 16S rRNA gene clone sequences and their most similar GenBank sequences which were not available in the Greengenes database at the time of analysis were identified from BLAST searches of sequences retrieved in this study and were then imported into the ARB software package (http://www.arb-home.de) []. [...] The Olsen corrected distance matrix was exported from the ARB program and all sequences were grouped into operational taxonomic unit (OTUs) by the furthest-neighbour algorithm Distance-based Operational Taxonomic Unit and Richness (DOTUR). DOTUR assigned sequences accurately to OTUs based on sequence data using values that are less than the cut off level []. A cluster with less than 3% substitutions in the phylogenetic tree was usually matched with the same species or relatives in GenBank as confirmed by the RDP Classifier results. In this study, a similar cut off of 97% was defined as an OTU. This same cut off was used for diversity indices and richness estimates that were calculated by DOTUR. In this study, the Shannon and Simpson diversity indices, and Chao and ACE richness estimates were calculated by DOTUR to estimate microbial diversity and richness. Bacterial species evenness was also calculated []. The Chao richness estimator curves were continuously calculated during the sequencing phase. When the estimator curve reaches a plateau, the sequencing effort was considered to be sufficient to provide an unbiased estimate of OTU richness, as proposed by Kemp & Aller []. Rarefaction curve was generated by plotting the number of OTUs observed against number of sasequences sampled. The P value generated from two tailed t-test was used to determine significance of difference between different parameters. […]

Pipeline specifications

Software tools BioEdit, ARB, DOTUR, RDP Classifier
Databases Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Infection, Catheter-Related Infections