Computational protocol: Fine Flounder (Paralichthys adspersus) Microbiome Showed Important Differences between Wild and Reared Specimens

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[…] Sequencing reads of 16S rRNA gene were processed using UPARSE () and analyzed using QIIME (). The quality of the reads was assessed using FastQC Software, and the reads were then filtered by quality and length by using the USEARCH algorithm. The resulting quality-filtered FASTA files were merged. Then, the sequences were trimmed to 140 bp, dereplicated, and sorted by abundance, and singletons were discarded. The reads were clustered into operational taxonomic units (OTUs) based on 97% identity using UPARSE with the “cluster_otu” command in USEARCH. Then, chimeric sequences were removed using the UCHIME algorithm. Taxonomic information was provided for each OTU with the “assign_taxonomy.py” QIIME script using the Ribosomal Database Project (RDP) as the reference database, with a confidence of 0.8. Representative sequences for each OTU were determined based on sequence frequency, and representative sequences were aligned using PyNAST algorithms. Phylogenetic relationships were determined based on representative sequence alignment using FastTree. Taxonomic assignments for each representative sequence were determined, and the above information was combined to construct the BIOM file using the “make_otu_table.py” QIIME script. These data are summarized in Supplementary Table . The graphics for the relative abundance of the composition of intestinal microbiota were performed in the R environment using the ggplot package. [...] Good’s coverage and alpha diversity indexes, including community diversity (Simpson and Shannon index), richness (Chao-1) and phylogeny-based metrics (PD Whole Tree), were calculated using the “alpha_diversity.py” QIIME script. The Mann–Whitney test was used to test the differences in alpha diversity (Shannon Diversity index, Simpson index, richness and PD Whole Tree) between the wild and aquaculture flounder using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). A p-value < 0.05 was considered statistically significant. Beta diversity measurements were investigated through a principal coordinate analysis (PCoA) performed on the phylogenetic beta-diversity matrix, obtained by weighted UniFrac analysis, using “beta_diversity.py” QIIME script. EMPeror was used to visualize the PCoA plots from weighted UniFrac metrics.Differential abundance of the bacterial components between wild and aquaculture flounder was assessed using linear discriminant analysis effect size (LEfSe), which considers both statistical significance and biological relevance (). LEfSe combines the standard tests for statistical significance (Kruskal–Wallis test and pairwise Wilcoxon test) with linear discriminate analysis (LDA) to estimate the effect size of each differentially abundant feature. The alpha value for the factorial Kruskal–Wallis test is 0.05, and the threshold for the logarithmic LDA score for discriminative features is 2.0. [...] To predict the metagenomes of each of the samples, a closed reference OTU-picking strategy based on the Greengenes database (version 13.5) was adopted, with a 97% sequence similarity threshold using the “pick_closed_reference_otus.py” QIIME script. The resulting OTU table, in biom format, was then used to generate inferred metagenomic data using PICRUSt () version 1.1.0 with the default parameters. The abundance values of each OTU were first normalized to their respective predicted 16S rRNA copy numbers. Predicted functional pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The accuracy of the predictions of the metagenomes was assessed by computing the NSTI (Nearest Sequenced Taxon Index), which is an index that indicates the relationship of the microbes in a particular sample to the bacterial genomes in a database (Supplementary Table ). The associated metabolic pathways were identified by employing HUMAnN2 (The HMP Unified Metabolic Analysis Network) with the default settings. The t-test was used to identify bacterial functional pathways that were differentially abundant in intestinal microbiota of wild flounder and aquaculture flounder. All p-values were corrected for an FDR of 0.05. FDR corrected p-values bellows 0.05 (FDR < 0.05) were considered significant (Supplementary Table ). […]

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