Computational protocol: Sorghum and wheat differentially affect caecal microbiota and associated performance characteristics of meat chickens

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Protocol publication

[…] DNA was isolated using the method of ; briefly, raw DNA extracts were obtained by initial lysis of caecal content in lysis buffer at 70 °C, followed by bead beating using a Precellys 24 instrument (Stretton Scientific Limited). This was followed by enzyme removal of RNA and protein and subsequent purification using Qiagen columns as per the supplier’s instruction. The V1-V3 region of the 16S rRNA gene was amplified (forward primer (), 5′ AGAGTTTGATCCTGG 3′; reverse primer W31 (), 5′ TTACCGCGGCTGCT 3′) as detailed by . Pyrosequencing was performed using a Roche/454 FLX+ instrument and Titanium chemistry kits according to the manufacturer’s instructions. Sff file processing was done using PyroBayes () and inspected for chimeric sequences using Pintail () and error corrected using Acacia (). Further trimming was done in QIIME () with sequence length 300–600 bases, no ambiguous sequences, minimum average quality score of 25 and maximum of 6 bases in homopolymer runs. OTU picking was done using Uclust (). Taxonomy was assigned using Blast against the GreenGenes database () and QIIME v.1.8 defaults. Additional taxonomic assignments for OTUs of interest was performed using EzTaxon database (). The complete dataset is available on MG-RAST database ( under library ID mgl538406. [...] Permutational analysis of variance (PERMANOVA) () was used to analyse differences between dietary treatments on each of the four performance measures (AME, FCR, BWG and FE) and also for differences in microbiota composition. The distance matrices were based on Euclidean distance (growth performance data) and both Weighted and Unweighted Unifrac distances (microbiota abundance data) (). The PERMDISP routine was used to test for the homogeneity of dispersions (based on mean distance to group centroids), to ensure that dispersions were constant among groups (). Principal Coordinates Analysis (PCO) was used to visualise between-groups differences in microbiota composition, based on both Unifrac distances. Analysis of variance (ANOVA) was used to test for differences in the diversity of microbial community between dietary treatments using four diversity measures (i.e., evenness, richness, Shannon’s and Inverted Simpson’s indices). ANOVA was also used to test for differences in the abundance of individual microbial taxa between dietary treatments. Redundancy discrimination analysis (RDA) was used to analyse individual associations between microbiota community composition and each of the performance measures in both dietary treatments. Pearson’s correlation tests were conducted to analyse the correlations between performance indicators. Spearman’s correlation test was used to analyse correlations between individual OTUs and each of the performance indicators. For all ANOVA, RDA and correlation analyses, microbiota abundance data was normalised and square root transformed to reduce variance heterogeneity and increase predictive power. PERMANOVA, PERMDISP and PCO analyses were carried out using PRIMER software (v.6.1.16) with the PERMANOVA+add-on (v.1.0.6). RDA, Pearson’s and Spearman’s correlation tests, and analyses of community diversity and taxonomic structure were performed in Calypso (v.5.8) (). […]

Pipeline specifications

Software tools PyroBayes, Acacia, QIIME, UCLUST, UniFrac, Calypso
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Sorghum bicolor, Triticum aestivum, Gallus gallus, Bacteria, Lactobacillus crispatus, Lachnospiraceae