Computational protocol: Analysis and comparison of the wolf microbiome under different environmental factors using three different data of Next Generation Sequencing

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Protocol publication

[…] First, operational taxonomical units (OTUs) were analyzed for each sample with a 97% sequence similarity cutoff value. Secondly, a summary of all taxonomic information was generated using RDP Classifier version 2.2. The phylogenetic relationship was elucidated using GraPhlAn. Finally, to standardize results, the lowest number of sequences from each sample was randomly selected and different data sets were observed to contain different homogeneous sequences. [...] Alpha diversity analysis facilitated the construction of a rarefaction curve and species accumulation boxplot. These were used to describe the number of OTUs and species as a function of sampling effort. Next, richness of the associated communities was compared based on the ACE estimator and the Chao1 estimator. Community diversity was subsequently analyzed using both the Simpson index and the Shannon index.Beta diversity analysis was used to determine microbiota composition diversity between the individuals using the linear Principal Component Analysis (PCA) model and the nonlinear Non-Metric Multi-Dimensional Scaling (NMDS) model. Wolf microbiota phylogenetic analysis was performed using an Unweighted Pair-group Method with Arithmetic Mean (UPGMA) and the associated phylogenetic trees were based on the Weighted Unifrac and Unweighted Unifrac values.To understand the correlation between parameters including attitude, pressure, climate and age, the Spearman correlation and the Mantel test correlation were calculated. LDA Effect Size (LEfSe) can search for a Metagenomic biomarker between the two groups and the biomarker is statistically significant (P < 0.05).We analyzed the three data sets using the Mothur (Version 1.36.1), Qiime (Version 1.7.0), Uparse (Uparse v7.0.1001), PyNAST (Version 1.2) and the following disgrams were made by R (R version 3.3.1). […]

Pipeline specifications

Software tools RDP Classifier, GraPhlAn, LEfSe, mothur, QIIME, UPARSE, PyNAST
Applications Phylogenetics, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Canis lupus familiaris, Homo sapiens
Diseases Pulmonary Fibrosis