Computational protocol: Characterising the interspecific variations and convergence of gut microbiota in Anseriformes herbivores at wintering areas

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Protocol publication

[…] Amplicons were sequenced using the Illumina Miseq platform at the Central South University, Changsha, China. Galaxy (http://mem.rcees.ac.cn:8080/) was used for sequencing data analysis unless otherwise noted. Raw data was sorted into independent files according to unique tags. After removal of tags and primers, pair-ended sequences were merged. Quality filtering included three steps: we discarded sequences (i) containing “N”; (ii) with quality score less than 20 (UPARSE pipeline and representative sequences of each OTU were generated simultaneously. Taxonomic assignment of representative sequences were performed with the Ribosomal Database Project (RDP) naïve Bayesian rRNA classifier at an 80% confidence level against a Greengenes reference taxonomy (Greengenes 13.8). Sequences identified as Achaea, Chloroplast and Mitochondria were excluded from subsequent analysis. The following alpha diversity metrics were calculated: Shannon diversity metric and observed OTUs metric. Rarefaction curves were generated with R “vegan” package. Representative sequences were then aligned using PyNAST with reference sequences from 16S Greengenes and a phylogenetic tree was constructed with FastTree.Resampled OTU table (10,000 sequences per sample) was used to calculate the α diversity (Shannon index and number of observed OTUs). UniFrac was employed to compare β diversity with the phylogenetic tree and the resampled OTU table. The Mann-Whitney test was used for univariate statistical analysis. All univariate statistical analysis was conducted using PRIMER 5 (PRIMER-E Ltd., Lutton, UK) and SPSS 21.0 (International Business Machines Corp., Armonk, NY). ANOSIM was performed to identify significant differences in gut microbiota structure using the method implemented in the R “vegan” package. The Mantel test was used to detect the correlation between diet composition and microbial community structures with the R “vegan” package. In this study, we used SIMPER in PRIMER 5 to filter out OTUs that contributed to 40% of dissimilarity between each two groups and these OTUs were selected as representatives which were used to produce a heatmap with “pheatmap” in R. [...] PICRUSt is a bioinformatics tool that uses marker genes to predict the functional content of microorganism. In this study, this method was employed to predict the potential functions of each sample based on 16S rRNA sequencing data. We used the KEGG database and performed closed reference OTU picking using the sampled reads against a Greengenes reference taxonomy (Greengenes 13.5). The 16S copy number was then normalized, molecular functions were predicted and final data were summarized into KEGG pathways. The differences in predicted molecular functions of bacterial communities among four groups were shown by PCA (principal component analysis) using R “vegan” package. ANOSIM was used to test whether the dissimilarity of gene abundance among the four groups was significant. All PICRUSt analyses were conducted online (http://mem.rcees.ac.cn:8080/root). […]

Pipeline specifications

Software tools UPARSE, PyNAST, FastTree, UniFrac, PHeatmaps, PICRUSt, vegan
Databases KEGG Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis, Transcriptome data visualization
Organisms Anser albifrons, Anser fabalis, Anser cygnoides, Hemisus marmoratus, Bacteria, Firmicutes