the soil bacterial community structure of a light rare earth tailings pond under different ecological restoration methods
Total soil DNA was extracted using the Fast DNATM SPIN Kit for Soil (MP Biomedicals, Solon OH, USA). PCR amplification of the V3 -V4 variable region of bacterial 16S rRNA gene was carried out using the 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') primers. PCR reactions mixture including 5TransStart FastPfu buffer 4 L, TransStart FastPfu DNA polymerase 0.4 L, 0.8 L (5 uM) of forward primer and reverse primer, respectively; 2 L of dNTPs (2.5 mM) and DNA Template (10 ng), ddH2O to a final volume of 20 L. The following PCR thermal cycling condition consisted of an initial denaturation step at 95 C for 3 min, followed by 27 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, and elongation at 72 C for 30 s, with final elongation step of 72 C for 10 min, finally stored at 4C (PCR instrument: ABI GeneAmp 9700). High-throughput sequencing of the PCR products was performed on an Illumina MiSeq PE 300 platform. Original sequencing quality-filtered by fastp version 0.19.6 and merged by FLASH version 1.2.7. The 300 bp reads were truncated at any site receiving an average quality score of <20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded, reads containing ambiguous characters were also discarded. Only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of overlap region is 0.2. Reads that could not be assembled were discarded. Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matching, 2 nucleotide mismatch in primer matching. Then the optimized sequences were clustered into operational taxonomic units (OTUs) using UPARSE 7.1 with 97% sequence similarity level. The most abundant sequence for each OTU was selected as a representative sequence. Species annotation of the obtained sequences was conducted using the Bayesian algorithm of the RDP classifier (Release 11.5 http://rdp.cme.msu.edu/). Bacterial sequences were obtained from the Silva (Release 148) database (Majorbio BioPharm Technology Co., Ltd., Shanghai).