Computational protocol: Elevated ground-level O3 negatively influences paddy methanogenic archaeal community

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Protocol publication

[…] The methanogenic archaeal 16 s rRNA gene data were processed using the Quantitative Insights Into Microbial Ecology (QIIME) 1.4.0-dev pipeline (; with the default parameters unless otherwise noted. In brief, sequences were quality trimmed (> 25 quality score and 200 bp in length), and assigned to soil samples based on unique 7-bp barcodes. Sequences were denoised and then binned into OTUs using a 97% identity threshold; the most abundant sequence from each OTU was selected as a representative sequence for that OTU. Taxonomy was assigned to methanogenic archaeal OTUs against a subset of the Silva 104 database ( OTU representative sequences were aligned using PyNAST, and chimera sequences were removed through QIIME. A phylogenetic tree was then constructed using FastTree2 to support phylogenetic diversity calculations.The richness of phylotypes was calculated to compare community-level methanogenic archaeal diversity at a single level of taxonomic resolution. We also estimated phylogenetic diversity using Faith's index, which provides an integrated index of the phylogenetic breadth across taxonomic levels. In this diversity analysis, 87,688 methanogenic archaeal sequences that passed QIIME's quality filtering were included. We obtained between 3,551 and 15,387 sequences per sample for all soil samples (mean = 7,307 and median = 6,801) (). Because an even depth of sampling is required for beta diversity calculations, we reduced the datasets to the lowest number available to correct for differences in survey effort between samples. Namely, we calculated both diversity metrics using a randomly selected subset of 3,500 sequences per soil sample. This approach allows us to compare general diversity patterns among sites even though it is highly unlikely that we surveyed the full extent of diversity in each community. The weighted pairwise UniFrac distances were calculated for community comparisons using QIIME and were visualized using non-metric multidimensional scaling plots as implemented in PRIMER v6. ANOSIM analyses were conducted using PRIMER v6. […]

Pipeline specifications

Software tools QIIME, PyNAST, FastTree, UniFrac
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Oryza sativa, Triticum aestivum
Chemicals Methane