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.