Computational protocol: Composted Cattle Manure Increases Microbial Activity and Soil Fertility More Than Composted Swine Manure in a Submerged Rice Paddy

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Protocol publication

[…] Raw sequences were processed using the FastX Toolkit and the QIIME pipeline (). Barcodes were recombined from paired-end reads, and forward reads were used for downstream analysis. All sequences were trimmed and low-quality sequences were removed. Quality filtering settings were as follows: minimum 30 quality scores over at least 75% of the sequence read; no ambiguous bases allowed; 1 primer mismatch allowed. Chimeras were further removed from the aligned 16S reads using a UCHIME algorithm (). The operational taxonomic units (OTUs) were identified based on 97% sequence similarity using a UCLUST algorithm () in QIIME and the Greengenes database was used to assign taxonomy to each OTU (). Additionally, we generated a species-level classification using ‘ClassifyReads’ a proprietary algorithm in MiSeq Reporter Metagenomics Workflow that provides species level classification for paired-end reads. This process involves matching short subsequences of the reads (called words) to a set of 16S reference sequences. The accumulated word matches for each read were used to assign reads to a particular taxonomic classification.Permutational multivariate analysis of variances (PERMANOVA) and analysis of similarity (ANOSIM) was carried out to test the significant differences in bacterial community composition. The response ratio (RR) was employed to illustrate the changes in bacterial relative abundance (). Mantel test, variation partitioning analysis (VPA) and canonical correspondence analysis (CCA) were performed to evaluate the linkages between the bacterial community and soil variables. To construct a CCA model, the predictor soil variables were selected based on their biological importance and p-values of single-factor CCA models and the variance inflation factor (VIF) criterion (VIF < 20). All the analyses were performed using functions in the Vegan package (v. 1.15-1) in R v. 2.8.1 (). Estimates of α-diversity such as Shannon diversity index, Margalef’s Richness and Pielou’s Evenness were performed using Mothur (v.1.28.0). Univariate analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test was used to test differences in soil biochemical properties among the treatments. […]

Pipeline specifications

Software tools FASTX-Toolkit, QIIME, UCHIME, UCLUST, mothur
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Bos taurus, Sus scrofa, Oryza sativa, [Clostridium] cellulolyticum, Pseudomonas stutzeri
Diseases Cattle Diseases
Chemicals Carbon, Nitrogen, Phosphorus