Computational protocol: Co-habiting amphibian species harbor unique skin bacterial communities in wild populations

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Protocol publication

[…] All sequence analyses were conducted using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline (). In brief, the QIIME pipeline removed low-quality sequences (that is, those sequences <200 bp in length, had a quality score <25, contained ambiguous characters, having an unreadable barcode or did not contain the primer sequence). Remaining sequences were clustered using CD-HIT (), using a minimum coverage of 99% and minimum sequence identity of 97%. A representative sequence was chosen for each phylotype by selecting the longest sequence with the highest number of hits to other sequences in the same phylotype. The representative sequences were aligned using PyNAST () and taxonomy was assigned using the RDP classifier (). A phylogenetic tree of all aligned sequences was constructed with FastTree () and used in all downstream phylogenetic community comparisons. Community similarity (that is, β-diversity) analyses were performed using the taxon-based Bray–Curtis dissimilarity metric, and the phylogenetic UniFrac metric: the Bray–Curtis metric examines taxon overlap, whereas the UniFrac metric is a phylogenetic approach based on the fraction of shared branch length between two communities within a phylogenetic tree (). Phylogenetic trees were constructed using FastTree (). All samples were rarified to 750 sequences per sample to remove sample heterogeneity, which impacts α- and β-diversity metrics (). […]

Pipeline specifications

Software tools QIIME, CD-HIT, PyNAST, RDP Classifier, FastTree, UniFrac
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Panthera pardus, Panthera tigris, Homo sapiens