Computational protocol: Weaned beef calves fed selenium-biofortified alfalfa hay have an enriched nasal microbiota compared with healthy controls

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Protocol publication

[…] During week 9 of the feeding trial, nasal swabs were collected from 5 or 6 weaned beef calves in each of the three treatment groups, by random selection of calves. One or two calves were selected from each pen (3 pens of 5 calves each per treatment group). Sterile, individually wrapped, polyester tipped applicators (Puritan®, Guilford, ME) were inserted approximately 10 cm into the nares, twirled to collect a mucosal swab, and then placed into individual sterile containers (10 mL, red topped, BD Vacutainer collection tubes; Becton Dickinson, Franklin Lakes, NJ) avoiding any contamination or contact with the wooden stick. Swabs in tubes were subsequently frozen at -80°C within 4 hours of collection. Negative control (sterile) swabs were similarly processed.Microbial DNA was extracted from nasal swab samples using MoBio Power soil DNA isolation kit (MoBio Laboratories, USA) as per the manufacturer’s instructions. The V4 region of the 16S rRNA gene was amplified with primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACVSGGGTATCTAAT-3′) at the MR DNA Laboratory (Shallowater, TX, USA) and sequenced on an Illumina MiSeq instrument at the MR DNA Laboratory (Shallowater, TX, USA). Raw sequence data was screened, trimmed, filtered, denoised and barcodes and chimera sequences were depleted from the dataset using QIIME v1.8 [] pipeline and UCHIME []. Operational taxonomic units (OTUs) were assigned based on at least 97% sequence similarity against the Greengenes reference database. For downstream analysis, sequences assigned as chloroplast, and mitochondria were removed. Sequences were rarefied to an even depth of 7,214 sequences per sample to account for unequal sequencing depth across samples. The sequences were deposited in NCBI SRA under accession number SRP090121. [...] Statistical analyses were performed using SAS version 9.2 []. Whole-blood Se concentrations were analyzed as repeated-measures-in-time using PROC MIXED. Fixed effects in the model were Se application rate (0, 45.0, and 89.9 g Se/ha), gender, baseline WB-Se (as linear covariate), time (after 2, 4, 6, and 8 weeks of feeding Se enriched hay), and the interaction between Se application rate and time. An unstructured variance-covariance matrix was used to account for variation of measures within calves. The unstructured variance-covariance matrix provided the most parsimonious variance-covariance matrix based on the lowest value by the Aikaike Information Criterion. To evaluate the effect of Se application rate, linear and quadratic contrasts were constructed. In addition, the linear response of the dependent variable Se forage content or WB-Se concentrations of beef calves to the independent variable Se fertilization rate were evaluated using univariate regression in PROC REG. Mean values were used for each pen, as pen was the experimental unit. Data are reported as least square means ± SEM. Statistical significance was declared at P ≤ 0.05 and a tendency at 0.05 < P ≤ 0.10.Nasal swabs were collected randomly from 5 or 6 calves in each of the three treatment groups and treated as individual samples. Differences in bacterial communities between the three groups were determined using the phylogeny based unweighted UniFrac distance metric and PCoA plots were generated with QIIME. ANOSIM (Analysis of Similarity) test within PRIMER 6 software package (PRIMER-E Ltd., Luton, UK) was performed on the weighted and unweighted UniFrac distance metrics to find significant differences in microbial communities between the groups.All the datasets were tested for normality with Shapiro-Wilk test (JMP Pro 11, SAS Software Inc.). Since most of the datasets did not meet the assumptions of normal distribution, non-parametric Kruskal-Wallis tests were performed. The resulting P-values were then adjusted for multiple comparisons using the Benjamini & Hochberg’s False Discovery Rate (FDR) for each taxonomic level, and an adjusted P < 0.05 was considered statistically significant (Benjamini and Hochberg, 1995). A Dunn’s post-test was used to determine which diets were significantly different. A significance value of P < 0.05 was selected for all statistical tests. Linear discriminant analysis effect size (LEfSe) was utilized to identify bacterial taxa that were differentially abundant between the healthy controls and calves fed Se enriched alfalfa hay. [...] To determine if there were any changes in microbial function in the nasal microbiota, PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was applied to make functional gene content predictions on the 16S rRNA gene sequencing data []. The results obtained with PICRUSt were analyzed using LEfSe to identify the significantly altered microbial functions between the groups. […]

Pipeline specifications

Software tools QIIME, UCHIME, JMP Pro, LEfSe, PICRUSt
Databases SRA Greengenes
Applications Miscellaneous, Phylogenetics, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Diseases Respiratory Insufficiency
Chemicals Selenium, Selenic Acid