Computational protocol: Seasonal Variability in Airborne Biotic Contaminants in Swine Confinement Buildings

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Protocol publication

[…] The extracted DNA was amplified using primers 338F (5′-XXXXXXXX-GTACTCCTACGGGAGGCAGCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) targeting the V3 region of bacterial 16S rRNA (‘X’ denotes 8-mer barcode sequence) . Paired-end sequencing was performed at Beijing Genome Institute (BGI) (Hongkong, China) using 2×150 bp Hiseq2500 (Illumina) according to the manufacturer's instructions. Library preparation, sequencing, and initial quality filtering were performed as described previously . The sequence data obtained by Illumina Hiseq2500 sequencing were processed using mothur . Paired-end sequences were assembled, trimmed, and filtered in mothur. Next, the sequences were aligned against a SILVA alignment ( Putative chimeric sequences were detected and removed via the Chimera Uchime algorithm contained within mothur . Sequences were denoised using the ‘pre.cluster’ command in mothur, which applies a pseudo-single linkage algorithm with the goal of removing sequences that are likely due to sequencing errors . All sequences were classified using the EzTaxon-e database ( , using the classify command in mothur at 80% Naïve Bayesian bootstrap cutoff with 1000 iterations. Sequence data were deposited in SRA at NCBI with the accession number of SRP039383. [...] Rarefaction curves and diversity indices were generated using mothur, with the bacterial phylotype (OTU) defined here at 97% threshold of 16S rRNA gene sequence similarity. Phylogenetic diversity (PD) was calculated as Faith's PD , the total phylogenetic branch length separating OTUs in each rarefied sample. To allow for robust comparisons among samples containing different numbers of sequences, phylotype richness and phylogenetic diversity were calculated based on samples rarefied to contain 15,909 sequences. To test for differences in relation to season on OTU richness, PD, 16S rRNA and TcR genes abundances, we used a t-test for normal data and the Wilcoxon rank-sum test for non-normal data. We used the same procedure to test whether the relative abundance of the most abundant phyla differed across seasons.To avoid including collinear variables in further analyses, we used a Spearman's correlation matrix and highly correlated (Spearman's r≥0.8) microclimatic variables were removed from further analysis. Non-metric multidimensional scaling (NMDS) was generated based on pairwise Bray-Curtis dissimilarities between samples using the vegan R package . The analysis of similarity (ANOSIM) function in the vegan R package with 999 permutations was used to test for differences in bacterial communities among the winter and summer season. The vectors of microclimate variables were fitted onto ordination space (Bray–Curtis NMDS) to detect possible associations between patterns of community structure and microclimate variables using the ‘envfit’ function of the vegan R package, and statistical significance was evaluated among 999 random permutations. Analyses for Venn diagram generation were performed using the mothur, and the Venn diagram was plotted using R package VennDiagram . Differentially abundant bacterial genera between the winter and summer seasons were identified using a parametric approach (Metastats) . All statistical analysis, graphs, and ordinations were produced using R version 3.0.2 . […]

Pipeline specifications

Software tools mothur, UCHIME, VennDiagram, Metastats
Applications Phylogenetics, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Sus scrofa, Homo sapiens
Chemicals Tetracycline