Computational protocol: Elevated atmospheric CO2 levels affect community structure of rice root-associated bacteria

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Protocol publication

[…] These genes were amplified as follows: 10 ng total bacterial DNA was used as a template in a final reaction volume of 50 μL including 0.1 μM of each primer and 2 U of Ex Taq DNA polymerase (Takara Bio, Shiga, Japan) with the universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 518R (5′-TTACCGCGGCTGCTGG-3′), containing the 454 FLX adaptors and a sample-specific multiplex identifier (Okubo et al., ; Ikeda et al., ). The cycling conditions were as follows: initial denaturation for 2 min at 94°C; 25 cycles of 30 s at 94°C, 30 s at 55°C, and 1.5 min at 72°C; and the final extension step of 8 min at 72°C. PCR products of the predicted size (~500 bp) were purified using the Wizard SV Gel and PCR Clean-Up System (Promega Japan, Tokyo, Japan). Sequencing was performed on 454 GS FLX+ (Roche Diagnostics K.K., Tokyo, Japan). The pyrosequencing reads were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package (Caporaso et al., ). The sequences were assigned to each sample according to the sample-specific multiplex identifier. Low-quality sequences shorter than 300 bp, with an average quality score lower than 25, with mismatching primer sequences, or with ambiguous bases (marked as “N”), were eliminated from downstream analyses. The forward and reverse primer regions were removed from the quality-filtered sequences. Potentially chimeric sequences were removed using the USEARCH6.1 software (Edgar, ). Potentially contaminated sequences classified as chloroplast, mitochondria, or unassigned by the RDP Classifier software (Wang et al., ) were removed. The remaining sequences were clustered into operational taxonomic units (OTUs) at 97% similarity using the pick_de_novo_otus command with default parameters. Principal coordinates analysis (PCoA) was performed on weighted and unweighted UniFrac distance matrixes (Lozupone and Knight, ) using a random sample of 2000 sequences for data normalization. For statistical testing, to determine the effects of the [CO2] elevation, rice genotype, and their interaction, permutational multivariate analysis of variance (PERMANOVA) was conducted on the UniFrac distance matrixes using the adonis function in the R software package vegan (http://vegan.r-forge.r-project.org/). The numbers of OTUs and Chao1 as well as Shannon, and Simpson's indexes were calculated with 10 replicates using a random sample of 2000 sequences for data normalization. Statistical analysis was performed on the mean values of 10 replicates to determine the effects of the [CO2] elevation, rice genotype, and their interaction using linear mixed model of the SPSS Statistics software, version 22 (IBM Japan, Tokyo, Japan). [CO2] and rice genotype were treated as fixed effects, while field and field × [CO2] were treated as random effects. The phylogenetic composition of the sequences was evaluated using the RDP classifier (Wang et al., ), with confidence levels of 80%. Statistical analysis was also performed on the relative abundance of each taxonomic group to determine the effects of the [CO2] elevation, rice genotype, and their interaction. […]

Pipeline specifications

Software tools QIIME, USEARCH, RDP Classifier, UniFrac, SPSS
Applications Miscellaneous, Phylogenetics, 16S rRNA-seq analysis
Organisms Oryza sativa
Chemicals Carbon, Methane