2.3 DNA extraction, PCR amplification, and sequencing
Microbial DNA was extracted from 0.5 g soil samples using Soil DNA Mini kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s protocol. The DNA preparation and sequencing library preparation were performed following the procedures described by Scholer and Vestergaard(Griffiths, Whiteley, O’Donnell, & Bailey, 2000). The V3-V4 region of the bacterial 16S rRNA gene were PCR amplified (95 °C for 2 min, followed by 27 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, with a final extension at 72 °C for 10 min) using the primer set 341F (5′-CCTACGGRRBGCASCAGKVRVGAAT-3′) and 806R (5′-GGACTACNVGGGTWTCTAATCC-3′). A six-base barcode was added to each library for further demultiplexing samples. All samples were subject to paired-end high throughput sequencing at regular sequencing depth. The BBW11 sample was also subject to deep sequencing to achieve at least 1 million reads per sample, in addition to regular depth sequencing. The Illumnina HiSeq 2500 (Illumina, Inc., San Diego, CA, USA) was used for sequencing.