3.7 Gene extraction, PCR amplicon, and sequencing analysis
Water samples obtained from the CBM well for gene sequencing were stored in an incubator filled with dry ice under a low temperature condition (0℃) while being transferred to the laboratory. The DNA for each sample was extracted with FastDNA SPIN Kit (MP Biomedicals). DNAs were measured by PicoGreen dsDNA Assay Kit (Life Technologies) and subsequently diluted to 3.5 ng/μl. The V3-V4 region of the bacterial 16S rRNA gene was amplified by degenerate PCR primers 341F: CCTACGGGNGGCWGCAG and 805R: GACTACHVGGGTATCTAATCC. Each sample was amplified in triplicate (together with water control) in a 30 μl reaction system, which contained 3 μl of diluted DNA, 0.75 U PrimeSTAR HS DNA polymerase, 1 x PrimSTAR buffer (Takara), 0.2 mM deoxyribonucleoside triphosphates (dNTPs) and 10 pM of barcoded forward and reverse primers. After an initial denaturation step at 98 °C for 30 s, the targeted region was amplified by 25 cycles of 98 °C for 10 s, 55 °C for 15 s and 72 °C for 60 s, followed by a final elongation step of 5 min at 72 °C. If there was no visible amplification from negative control (no template added), triplicate PCR products were mixed and purified using an AMPure XP Kit (Beckman Coulter). The purified PCR products were measured by Nanodrop (NanoDrop 2000C, Thermo Scientific), and diluted to 10 ng/μl as templates for the second step of the PCR. All samples were amplified in triplicate with second-step primers, using identical conditions to the first step of the PCR but with eight cycles.
Technical replicates of each sample were combined and run on a 1.2% (w/v) agarose gel, and the bacterial 16S rRNA gene amplicons were extracted using a QIAquick Gel Extraction Kit (Qiagen). DNAs were subsequently measured with a PicoGreen dsDNA Assay Kit (Life Technologies) and 10 ng of each sample were mixed. Final amplicon libraries were purified twice using a Agencourt AMPure XP Kit (Beckman Coulter) and subjected to a single sequencing run on the HiSeq 2500 platform (Illumina Inc).
Bioinformatics analysis on 16S rRNA gene profiling. The 16S rRNA gene sequences were processed using QIIME v.1.9.1 and USEARCH v.10.0. The quality of the paired-end Illumina reads was checked by FastQC v.0.11.5 and processed in the following steps by USEARCH: joining of paired-end reads and relabeling of sequencing names; removal of barcodes and primers; filtering of low-quality reads; and finding non-redundancy reads.
Unique reads were clustered into OTUs with 97% similarity. OTUs were aligned to the SILVA database to remove sequences from chimera. The OTU table was generated by USEARCH. The taxonomy of the representative sequences was classified with the RDP classifier. Functional annotations of prokaryotic taxa were carried out using Picrust2 against the KEGG databases.
Three complementary non-parametric multivariate analyses, non-parametric multivariate analysis of variance (Adonis), analysis of similarity (ANOSIM), and the multi-response permutation procedure (MRPP;), were used to test the differences in soil microbial communities between warming and control treatments. The difference of the C-N-S function genes was detected by Welch’s t-test with FDR correction in STAMP. The heatmaps were drawn by the “pheatmap” package.
A maximum likelihood phylogeny of main C-N-S microbes was generated from the aligned RDP sequence using Iqtree. All phylogenetic trees were edited in Itol.
To acquire the best discriminant performance of C-N-S microbes across runoff and stagnant area, we classified the abundances of bacterial taxa using the “randomForest” package. Cross-validation was performed by the rfcv function for selecting appropriate features. The varImpPlot function was used to show the importance of features in the classification.