Computational protocol: Diversity and Composition of Sulfate-Reducing Microbial Communities Based on Genomic DNA and RNA Transcription in Production Water of High Temperature and Corrosive Oil Reservoir

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[…] The total number of bacterial 16S rRNA and dsrA gene transcripts was determined for each sample of this study. The quantification was performed using a CFX96 thermalcycler (Bio-Rad, United States) with the SYBR Green system. The reactions for the two genes were performed using 25 μl reaction volume with 12.5 μl SYBR Green Master Mix (Takara, Japan), 9.5 μl of ddH2O, 0.5 μl of each PCR primers, and 2 μl of cDNA sample. The 16S rRNA gene transcript was amplified using the following primer sets: 338F (5′-ACTCCTACGGGAGGCAG-3′) and 805R (5′-GACTACCAGGGTATCTAATCC-3′) (). The amplification was carried out as follows: an initial denaturation step of 4 min at 94°C, 40 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s and extension at 72°C for 1 min. The dsrA gene transcripts were amplified with the same primers () as described above for the clone library. The PCR reaction conditions were as follows: initial denaturation at 94°C for 2 min; 40 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 30 s, and a final extension step at 72°C for 5 min. Annealing temperature was experimentally optimized to maximize the specificity of amplification (data not shown). The specificity of this primer pair was checked using the ProbeCheck against dsrAB database which contains 7,695 publicly available partial (6403) and full length (1,292) sequences (). The standard curves for RT-qPCR were generated through 10-fold serial dilutions of plasmids carrying the specific target gene inserts (each run in triplicate). No-template negative controls were used to check for cross contamination. The size of the PCR product was checked with agarose gel electrophoresis. Melting curve (range: from 65 to 95°C) analysis was also conducted following each assay to confirm the specificity of the primer pairs. Amplification efficiency was calculated based on the respective standard curve. Results were expressed in gene copy numbers per ml of production water. [...] aprA and dsrA diversity calculations were performed with the EstimateS () including Shannon and reciprocal of Simpson index. Sampling coverage was evaluated by using non-parametric richness estimators ACE (abundance-based coverage estimator) (), Chao1 (; ) and Coverage. Rarefaction curves were generated using GraphPad Prism 6. Bubble plots were made using the software R v3.2.4 () with the ggplot2 () and reshape2 () package. The Pearson correlation coefficients (r) between the gene copy numbers and environmental variables and Spearman′s rank correlations (rs) between the relative abundance of individual taxa and environmental factors were investigated via in R software with Hmisc package (). Differences in total and active microbial community composition were visualized through Non-metric Multi-Dimensional Scaling (NMDS) using Bray–Curtis similarity index by PAST software (version 3.09) (). OTU abundances for bacteria and archaea underwent square-root transformation to reduce the stress. Two-way PERMANOVA was performed to tests the differences in bulk and active bacterial and archaeal communities and location of community. The Bray–Curtis dissimilarity was calculated for each bacterial and archaeal genomic and active community based on OTU abundance and analyzed via PERMANOVA (999 permutations) in the PAST. Canonical correspondence analysis (CCA) was performed in CANOCO 4.5 for windows to identify the relationships between sulfate-reducing community structure and environmental parameters (). […]

Pipeline specifications

Software tools probeCheck, Ggplot2
Applications Miscellaneous, qPCR
Chemicals Adenosine Phosphosulfate