Computational protocol: Dynamics of Soil Bacterial Communities in Response to Repeated Application of Manure Containing Sulfadiazine

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Protocol publication

[…] The two data sets acquired by the forward and reverse primers were analyzed separately to avoid the systematical bias of individual primers. The analyses were mainly performed according to Ding et al. . Only those sequences with a length above 200 bp after removing the barcode, primer and unpaired regions were subjected to further analysis. The unpaired regions were truncated based on a standalone BlastN analysis against a bacterial database described by Pruesse et al. . Sequences were grouped into operational taxonomic units (OTUs>97% sequence identity) using software package mothur (v1.14.0). The classification of sequences was performed using software RDP MultiClassifier at >80% confidence . A taxonomic OTU report with each row representing one OTU containing taxonomic positions (from phylum to genus) and the number of sequences for each sample was constructed based on the OTU assignment and on the classification.Analyses based on the taxonomic OTU report were done with R (version 2.14). To allow the comparison of samples with different number of reads, all algorithms selected rely on relative abundance of taxonomic groups. To check whether the forward and reverse primers revealed a similar taxonomic composition or not, the percent of Bray-Curtis similarity of taxonomic composition between two data sets was calculated for each sample based on the percent of classified taxa at phylum, class, order, family, and genus level using the R add-on package “vegan”. To evaluate the influence of repeated application of manure containing SDZ on the bacterial community compositions, the pairwise Pearson dissimilarities between S0 and S10 or between S0 and S100 for day 193 were calculated. To compare the microbial community composition between samples, non-metric multidimensional scaling (NMDS) analysis was carried out based on the Pearson dissimilarity matrix using R package MASS. The 3-dimensional plots of NMDS analysis were created with the R package “rgl” in conjunction with the Linux command “import”. Tukey's honest significance tests under a generalized linear model via a logistic function for binomial data with the package multcomp was applied to identify the discriminative taxa between treatments using a Bonferroni adjusted p value<0.05. […]

Pipeline specifications

Software tools BLASTN, mothur, multcomp
Application GWAS
Organisms Homo sapiens
Chemicals Sulfadiazine