Computational protocol: Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies

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Protocol publication

[…] Bioinformatic analysis was performed using Mothur software (version 1.34.4) []. Good-quality sequences were classified as "operational taxonomic units" (OTUs), grouped at 97% similarity and classified based on the Ribosomal Database Classifier (RDP, March 2012) []. Chimera detection was performed using Uchime [].Sub-sampling was performed based for the sample with the minimum number of reads to reduce the errors in non-uniform sequences. To ensure sub-sampling representation, Good's coverage and rarefaction curves that represented the number of OTUs by the number of reads for each sample were evaluated. The diversity of each sample was estimated by calculating the inverse of Simpson’s index, and richness was calculated using Catchall software []. Bar graphs representing the relative abundance of the main phyla and genera present in each group at the different time points were generated, and the Steel-Dwass test was used to compare relative abundances between groups, controlling for multiple comparisons error. The ANOVA test was used to compare the results from Catchall and the inverse of Simpson’s index calculated for each group (p < 0.05 significance level).The similarity between the bacterial populations in each sample was compared using a distance matrix in PHYLIP format to calculate the Yue & Clayton coefficient, which takes into account the bacterial richness and evenness (community structure), as well as the Classic Jaccard coefficient, which takes into account only bacterial richness (membership). The calculations were visually represented by dendrograms that were built using Figtree software (version 1.4.0, http://tree.bio.ed.ac.uk/). Principal coordinate analysis (PCoA) was also performed to compare sample similarities in 3 dimensions using the JMP software (SAS Institute Inc.). The similarity between community structure and membership found in samples from each group was compared using the Parsimony test and by calculating the analysis of molecular variance (AMOVA). The parsimony method is a generic test that describes whether two or more communities have the same structure.Significant differences over time (pre- vs. post-conditioning) of the energy intake, concentrate, body weight, and plasma and serum biomarkers (lactate, pH, AST, and CK) were assessed using ANOVA followed by the Holm-Sidak test. Student's t-test for paired samples was used to determine the impact of acute exercise (baseline IETs and fatigue). Statistical power-analysis test was performed. The Pearson correlation was used to estimate the relationship between Catchall results, the inverse of Simpson’s index, CK and AST enzymatic activities, lactate concentration, and plasma and fecal pH. All analyses were performed at the 5% significance level. […]

Pipeline specifications

Software tools mothur, UCHIME, PHYLIP, FigTree
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Equus caballus, Homo sapiens, Rattus norvegicus