Computational protocol: Soil Bacterial Community Was Changed after Brassicaceous Seed Meal Application for Suppression of Fusarium Wilt on Pepper

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Protocol publication

[…] For each soil DNA, the 16S universal bacterial primer set 27F (5′-AGR GTT TGA TCM TGG CTC AG-3′) and 519R (5′-GTN TTA CNG CGG CKG CTG-3′) was used for amplifying the ∼ 500 bp region of 16S rRNA genes. To resolve different samples, an 8-bp barcode was fused to the forward 27F primer. A HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA, United States) was used for PCR under the following conditions: 94°C for 5 min, followed by 32 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and a final elongation step at 72°C for 5 min. The PCR products was visualized and confirmed on a 1.8% agarose gel. All PCR amplicons from different samples were then purified using Agencourt AMPure beads (Agencourt Bioscience Corp., Beverly, MA, United States). The concentration of each purified PCR amplicon was determined using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, Delaware, United States). Equimolar concentrations of the amplicons were then merged into a single tube and subjected to pyrosequencing at Molecular Research Laboratory (Shallowater, TX, United States) using 454 GS FLX titanium technology (454 Life Sciences, Branford, CT, United States). The amplicons were sequenced in the forward direction.The 454 pyrosequencing data were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) Pipeline. Specifically, the low-quality sequence reads (reads lengths < 150 bp, ambiguous bases > 0, homopolymers > 6, barcode mismatches, and average quality scores < 25) were discarded, and the 8-bp barcode was examined to distribute the sequences to proper samples. Then, chimeric sequence reads were identified and filtered using the Uchime algorithm based on a chimera-free reference database () through the Usearch tool. The operational taxonomic units (OTUs) were generated at a 97% sequence-similarity level (). A representative sequence of each OTU was aligned with the PyNAST tool (). The taxonomic identification of OTUs was obtained using the RDP Classifier with RDP as the reference database ().To standardize sampling efforts and to bring the pyrosequences of each sample onto a common scale, all samples were rarefied to an equal sequence depth (866 sequences per sample in this study). The relative abundance of all the phylotypes at each taxonomic level (phylum, class, order, family, and genus) was then summarized. Principal coordinate analysis (PCoA) was performed using the subsampled sequence data to determine the differences in the microbial community structures based on the weighted UniFrac distances, a method that accounts for the phylogenetic relationship between sequences and is thus more powerful than taxon-based measures (; ). Samples were clustered using UPGMA (unweighted pair group method with arithmetic mean) on weighted UniFrac distances. Three different complementary non-parametric analyses for multivariate data (), namely, the analysis of similarities (ANOSIM) (), non-parametric multivariate analysis of variance using distance matrices (adonis) (), and a multi-response permutation procedure (MRPP) (; ), were used to test for differences in the community structure between treatments. Weighted UniFrac distances were exploited for ANOSIM, Adonis, and MRPP analyses, and a Monte Carlo permutation was used to determine the statistical significance. ANOSIM, adonis, and MRPP were performed using the “vegan” package in the R environment.For the relative abundance data of each of the taxa, the linear discriminant analysis (LDA) effect size (LEfSe) method was used to test significant differences between treatments. Furthermore, Pearson correlation analysis was performed to understand the possible relationship between the bacterial taxa and the disease index. The Pearson correlation analysis were processed with the software SPSS. […]

Pipeline specifications

Software tools QIIME, UCHIME, USEARCH, PyNAST, RDP Classifier, UniFrac, LEfSe
Applications Phylogenetics, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Bacteria, Camelina sativa, Brassica juncea