16S rRNA of cave sediment metagenome
DNA was isolated with a MoBio Power SoilDNA isolation kit (USA). 16S rRNA amplicon metagenomic analysis was based on 454 pyrosequencing diversity assay (bTEFAP, www.mrdnalab.com) to assess the bacterial diversity using barcoded 16S universal eubacterial primers (forward, 27Fmod 5 AGR GTT TGA TCM TGG CTC AG 3, reverse, ill519Rmod 5 GTN TTA CNG CGG CKG CTG 3). A single-step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, USA) was applied under the following conditions: 94 C for 3 minutes, followed by 28 cycles of 94 C for 30 seconds, 53 C for 40 seconds and 72 C for 1 minute, with a final elongation at 72 C for 5 minutes. Following PCR, all amplicon products from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, USA). Samples were sequenced utilizing Roche 454 FLX titanium instruments (USA).