Computational protocol: Methanogenic Community Was Stable in Two Contrasting Freshwater Marshes Exposed to Elevated Atmospheric CO2

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Protocol publication

[…] For methanogens, the primer set 1106F and 1378R was used to amplify approximately 280 bp of methanogenic archaeal 16S rRNA gene fragments (). The forward primer contained a sample-identifying barcode unique to each sample. PCR amplifications were performed in 50 μl reaction mixtures containing approximately 40 ng of template DNA, 25 μl Phusion High-Fidelity PCR Master Mix (New England Biolabs), 20.5 μl H2O, 0.5 μl 1% bovine serum albumin (BSA), and 0.2 μM of each primer. Thirty cycles (95°C for 45 s, 56°C for 45 s, and 72°C for 60 s) were performed with a final extension at 72°C for 7 min. PCR products were mixed in equidensity ratios. Then, the mixed PCR products were purified with the Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, United States) following the manufacturer’s recommendations. The library quality was assessed on the Qubit 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 systems. Finally, high-throughput sequencing of the methanogenic archaeal 16S rRNA genes was carried out on the Illumina HiSeq2500 platform at the Novogene (Beijing, China), and 250 bp paired-end reads were generated.The high-throughput sequencing data were processed using the Quantitative Insights Into Microbial Ecology (QIIME) 1.7.0-dev pipeline () using default parameters, unless otherwise indicated. In brief, low quality sequence reads were trimmed and the barcode was examined to assign the multiplexed reads to samples. Next, the sequences were compared with the reference database () using the UCHIME algorithm to detect chimeric sequences, and the chimeric sequences were removed. Sequences with ≥97% similarity were assigned to the same OTUs using the Uparse software (Uparse v7.0.1001) (), and the most abundant sequence from each OTU was selected as a representative sequence for that OTU. Taxonomy was assigned to methanogenic archaeal OTUs against the GreenGene Database () using a RDP classifier (). To remove the heterogeneity of the number of sequences per sample, the sequences were rarefied prior to the calculation of the alpha diversity statistics. Alpha diversity was assessed by calculating Chao1 (), Observed species, Phylogenetic Diversity (PD), and Shannon metrics in QIIME. All sequences have been deposited in the DNA Data Bank of Japan under the accession number DRA005365. […]

Pipeline specifications

Software tools QIIME, UCHIME, UPARSE, RDP Classifier
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Oryza sativa
Chemicals Carbon Dioxide, Methane