Computational protocol: Bacterial Communities Associated with Atherosclerotic Plaques from Russian Individuals with Atherosclerosis

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Protocol publication

[…] The 16S data were further analyzed using the bioinformatics pipeline QIIME, version 1.9.1 []. FlowClus was used as a systematic approach to filter and denoise the received reads []. Sequences that were shorter than 150 bp were removed from the pyrosequencing-derived datasets, and read end sections of 50 bp dropping below the quality score threshold of 20 were trimmed. Probable chimeric sequences were identified using an implemented USEARCH 6.1 [] and further removed. Sequences were clustered into operational taxonomic units (OTU) (at least five reads for an OTU) based on the 97% identity threshold (reference-based OTU picking followed by de novo OTU clustering of sequences). For the taxonomic classification, the latest Greengenes reference (release v.13_8) [] and RDP Classifier 2.2 were used. Observed OTU numbers, Simpson, Chao 1 and Shannon diversity indices along with the phylogenetic diversity were calculated as the indicators for alpha diversity. To determine beta diversity among all atherosclerotic plaque samples, beta diversity matrices were performed using UniFrac metrics implemented in QIIME with following principal coordinate analysis (PCoA). Heat map of the relative abundances of OTUs in individual atherosclerotic plaques was created with R package. Correlations between the abundances of different taxa in atherosclerotic plaque samples and various clinical values were analyzed using R Hmisc package (based on the Spearman’s rank correlation coefficient). The obtained pyrotag sequences were deposited under the EMBL-EBI accession number PRJEB12836 (http://www.ebi.ac.uk/ena/data/view/PRJEB12836). In addition, GraphPad Prism 5 as well as Microsoft Excel 2013 were used for data analyses and their interpretation. […]

Pipeline specifications

Software tools QIIME, FlowClus, USEARCH, RDP Classifier
Databases Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Bacteria, Homo sapiens
Diseases Cardiovascular Diseases, Atherosclerosis
Chemicals Alanine, Cholesterol