Computational protocol: The impact of diet on the composition and relative abundance of rumen microbes in goat

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Protocol publication

[…] The primer sequences and multiplexing barcodes were trimmed. The poor/low quality sequences including those with uncertain nucleotides, continuous three nucleotides with Q value less than 20, unmatched barcode sequences were discarded. The obtained sequences were clustered using Uparse7.0 into operational taxomonic units (OTUs) based on 97% similarity. Chimeras were removed using UCHIME 4.2.4 []. The most abundant sequence was selected as the representative for each OTU, and then aligned against the Greengenes database (http://greengenes.lbl.gov) using PyNAS []. Representative sequences were taxonomically classified using the ribosomal database project (RDP) taxonomic database. Calculations for rarefraction curves, the estimated maximum number of OTUs, Shannon-wiener index and chaoI index were performed using QIIME version 1.8.0 []. The current % coverage was calculated by dividing the observed number of OTUs by the maximum number of OTUs estimated. The Weighted UniFrac distance metric was calculated to compare the phylogenetic similarity between the bacterial communities. Principal co-ordinate analysis (PCoA) cluster plot was performed using R version 3.1.0 for visualization. All Illumina sequences data in this study were deposited to the SRA of the NCBI database under BioProject PRJNA290546. [...] The relative abundance of microbes was log-transformed [] and the unpaired two-tailed t-test was performed using the SPSS Statistics software v. 19.0 (IBM, Armonk, NY, USA) to assess whether there was a significant difference between CF and AF. Significance level was set at p<0.05. […]

Pipeline specifications

Software tools UPARSE, UCHIME, QIIME, SPSS
Applications Miscellaneous, Phylogenetics, 16S rRNA-seq analysis
Organisms Capra hircus