Computational protocol: The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media

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Protocol publication

[…] Aliquots of URT amplicon samples were standardized to the same concentration and pooled into 5 different sequencing runs according to individual barcode primers for the 16S rRNA gene. Final equimolar libraries were sequenced using the MiSeq reagent kit v2 (300 cycles) on the MiSeq platform (Illumina, Inc., San Diego, CA). The generated 16S rRNA gene sequences were processed through the open source software pipeline Quantitative Insights Into Microbial Ecology (QIIME) version 1.7.0-dev. Sequences were filtered for quality using established guidelines. Sequences were binned into OTUs based on 97% identity using UCLUST against the Greengenes reference database, May 2013 release. Low-abundance clusters were filtered and chimeric sequences were removed using USEARCH. The representative sequences for each OTU were compared against the Greengenes database for taxonomy assignment, and only full-length, high-quality reads (−r = 0) were used for analysis. Additionally, we generated a species-level OTU table using the MiSeq Reporter Metagenomics Workflow. The MiSeq Reporter classification is based on the Greengenes database ( and the output of this workflow is a classification of reads at multiple taxonomic levels: kingdom, phylum, class, order, family, genus, and species.Shannon and Chao1 indexes output were generated by QIIME pipeline. Before estimating the Shannon and Chao1 indexes, all sample libraries were rarefied to an equal depth of 10,000 sequences using QIIME. Chao1 and Shannon indexes, total number of reads, and the log of the 16S rDNA copy number (total bacterial load) were analyzed using repeated measures ANOVA by general linear models fitted in JMP Pro 11 (SAS Institute Inc., Cary, NC). Dunnett’s multiple comparisons procedure was performed to compare the mean number of reads, Shannon index and Chao 1 index of each disease status (otitis, pneumonia, and pneumonia-otitis combined) against the healthy samples within each day of data collection (days 3, 14, 28, and 35).Correlations between total bacterial load and alpha-diversity indexes (Shannon and Chao 1 indexes) were assessed using simple linear regression in JMP Pro 11 software (SAS Institute Inc.).The relative abundances of microbial phyla and genera types in URT samples of calves at ages 3, 14, 28, and 35 within each health status were compared using general linear models fitted in JPM Pro 11 (SAS Institute Inc.). Dunnett’s multiple comparisons procedure was used to compare the mean relative abundance of the most abundant bacterial phyla and the genera of each disease status (otitis, pneumonia, and pneumonia-otitis combined) against the healthy samples within each day of data collection (days 3, 14, 28, and 35). Differences with a value of P ≤ 0.05 were considered significant and those with a value of 0.05 < P ≤ 0.10 were considered tendencies.Descriptive statistics for birth weight (BW) and average daily gain (ADG) were determined according to health status by using a general linear model (ANOVA) with JMP Pro 11 (SAS Institute Inc.). In total, 174 calves were enrolled in this study. Number of calves, disease incidence, mortality, BW and ADG (calculated by subtracting BW from the weaning weight and then dividing by days of life at weaning) during the pre-weaning period are presented in . Average age (days in life) at first diagnosis of pneumonia, otitis and pneumonia_otitis combined was assessed by using Distribution platform offered by JMP Pro 11 (SAS Institute Inc.). […]

Pipeline specifications

Software tools QIIME, UCLUST, USEARCH, JMP Pro
Databases Greengenes
Applications Miscellaneous, Metagenomic sequencing analysis
Organisms Bos taurus, Bacteria
Diseases Bacterial Infections, Mycoplasma Infections, Otitis, Pneumonia, Respiratory Insufficiency