Computational protocol: Consistent responses of the microbial community structure to organic farming along the middle and lower reaches of the Yangtze River

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Protocol publication

[…] Amplicons were sent out for pyrosequencing on the Illumina platform. The Majorbio-Shanghai. Illumina MiSeq PE300 was used to sequence the samples. After sequencing, the Trimmomatic was used to process the raw sequences, and the PE reads were overlapped by flash to assemble the final V4-V5 tag sequences. The uparse method (version 7.1) on the Usearch software platform (version 7.1 was employed to assign operational taxonomic units (OTUs) to the 16S rRNA at a cutoff level of 3%. The Uchime (version 4.2.40) method was used to correctly detect and remove chimeras produced by PCR. To compare relative differences between the samples, a randomly selected subset of 41105 sequences per sample was performed for downstream analyses. [...] An analysis of variance (ANOVA) was performed to evaluate the main effects of the agricultural system, farm location, crop type and the interactions of these factors on the soil physical and chemical analyzed. Based on the OTU results, the rarefaction curve and Shannon index curve were analyzed using Mothur (version v.1.30.1), and the richness estimators (Chao1, Abundance-based and Coverage Estimator (ACE)) and diversity indices (Shannon and Simpson) were determined using Mothur. Principal component analysis (PCA) and redundancy analysis (RDA) was performed with the rda() function in the vegan package in R (Version 3.0.2, vegan package). A Venn diagram was generated using R (Version 3.0.2, venn Diagram package). LEfSe was used to find indicator bacterial groups specialized within the organic and conventional types of samples. Statistical analysis was conducted using SPSS 13.0. […]

Pipeline specifications

Software tools Trimmomatic, UPARSE, USEARCH, QIIME, UCHIME, mothur, LEfSe
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Escherichia coli, Oryza sativa, Bacteria
Diseases Pulmonary Fibrosis