Computational protocol: The gut microbiome and degradation enzyme activity of wild freshwater fishes influenced by their trophic levels

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Protocol publication

[…] All sequences have been deposited in the NCBI’s Sequence Read Archive (SRA accession number to be provided upon acceptance). FLASH software was used to merge Pairs of reads from the original DNA fragments when the original DNA fragments are shorter than twice the length of reads. Raw data were filtered using the open-source software system Quantitative Insights into Microbial Ecology (QIIME) quality filters. Then we used UPARSE pipeline to pick operational taxonomic units (OTUs) at an identity threshold of 97%. We picked a representative sequences for each OTU and used the RDP classifier tool to assign taxonomic data to each representative sequence.In order to estimate individual hosts’ microbial alpha diversity, the rarefaction curves were generated based on metrics and the number of operational taxonomical units (OTUs) present in the samples was determined (Chao1 metric and shannon index). To minimize the biases caused by sequencing effort differences, equal numbers of sequences were used. The values were summarized per species of fish as the means and standard deviations ( ± σx). A 97% sequence identity of the 16S rRNA gene was used to determine OTUs and calculated the richness of Chao1 and indexes of Shannon diversity in species-level.Core gut microbiota for each fish species was defined and analyzed by indentified the shared OTU among three replicates. The shared and unique OTUs among the four trophic levels were also represented by a Scale-Venn diagram using eulerAPE (http://www.eulerdiagrams.org/eulerAPE/). The gut microbiota, beta diversity and taxon composition were analyzed by QIIME for calculating both weighted and unweighted UniFrac.To better understand the forces that shape the fish gut composition, the eight fish species were sampled from the same area and assigned to four different diet categories (herbivorous, carnivorous, omnivorous or filter-feeding). The unweighted UniFrac phylogenetic distance metric was analyzed by using a Principal Coordinate Analysis (PCoA) and Unweighted Pair Group Method with Arithmetic mean (UPGMA) Clustering. To explore the metabolic activity of the bacterial communities found on the gut contents of different trophic level fish species, a bioinformatics tool PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to generate the KEGG (Kyoto encyclopedia of genes and genomes) pathway. The functions were categorized at levels 2 and 3. […]

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