Computational protocol: Atmospheric Deposition-Carried Zn and Cd from a Zinc Smelter and Their Effects on Soil Microflora as Revealed by 16S rDNA

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Protocol publication

[…] Total bacterial 16S rDNA gene was amplified using the bacterial universal primers 515f forward (5′-GTGCCAGCMGCCGCGGTAAT-3′) and barcoded-806r reverse (5′-GGACTACHVGGGTWTCTAA-3′), which target a broad diversity of bacteria with few biases against particular groups according to a standard protocol. The PCR mixture (final volume 50 μL) contained 20 μL Premix Ex Taq (Takara Biotechnology), 0.4 μL of each primer (10 μM), 4 μL of five-fold diluted template DNA (1–10 ng) and 25.2 μL-sterilized water. Polymerase chain reaction (PCR) was performed with an initial 1 cycle of denaturation (3 min at 94 °C), six touchdown cycles of 45 s at 94 °C, 60 s from 65 °C to 58 °C, 70 s at 72 °C, followed by 22 cycles of 45 s at 94 °C, 60 s at 58 °C, 60 s at 72 °C with a final elongation of 72 °C for 10 min. Finally, the PCR products were purified by Wizard SV Gel and PCR Clean-up system (Promega, San Luis Obispo, CA, USA). The concentrations of the PCR products were fluorometrically quantified by the Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA) before being sequenced on the Miseq platform (Illumina, San Diego, CA, USA), at Novogene, Beijing, China. Raw sequences were processed in QIIME 1.7.0. Sequences were quality trimmed and clustered into operational taxonomic units (OTUs) at a 97% identity threshold using UCLUST. Representative sequences from individual OTUs were then aligned against the Green-genes core set using PyNAST. Taxonomic assignment was carried out with the RDP Classifier. The principal coordinate analysis (PCoA) was used to visualize the Bray-Curtis dissimilarity matrices based on the 97% OTU level across different copper concentrations. Diversity was characterized by calculating richness (OTU numbers, Shannon index) and evenness (Gini coefficient). The Gini coefficient (ranging from 0 to 1) is a value to assess the specific degree of evenness, and a higher Gini coefficient indicates lower evenness of a community. The Gini coefficient was calculated by performing ineq package in R3.0.2 software ( […]

Pipeline specifications

Software tools QIIME, UCLUST, PyNAST, RDP Classifier
Application 16S rRNA-seq analysis
Organisms Bacteria
Diseases Escherichia coli Infections
Chemicals Zinc