title: h
design: Triplicate PCR reactions were performed for each sample. The forward primer (5’- CTATGCGCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG- 3’) contained the 454 Life Sciences primer B sequence, the broadly conserved bacterial primer 27F, and a two-base linker sequence (“TC”). The reverse primer (5’- CGTATCGCCTCCCTCGCGCCATCAGNNNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’) contained the 454 Life Sciences primer A sequence, a unique 10-nt barcode used to tag each PCR product (designated by NNNNNNNNNN and provided by Roche), the broad-range bacterial primer 338R, and a “CA” linker sequence inserted between the barcode and the rRNA primer. Each 25 µl reaction contained 0.2 µM forward and reverse primers, 3 µl template DNA, and 2.5 ul 10X PCR buffer plus Mg2+ (TaKaRa), 2.0 ul dNTP (2.5 mM each) (TaKaRa), 0.75 ul DMSO (100%), 0.25 ul TaKaRa TaqTM (5 U/µl). All dilutions were carried out using sterile ddH2O. PCR reactions were assembled within a PCR hood in which all surfaces and pipettes had been decontaminated with 30 min autoclaving at 120? and UV irradiated for 30 minutes. Thermal cycling was 95°C for 10 minutes, followed by 30 cycles of 95°C for 30 seconds, 52°C for 30 seconds, and 72°C for 90 seconds, with a final extension of 10 minutes at 72°C. Replicate amplicons were pooled and visualized on 1.5% agarose gels using EB stain in 0.5X TE. Amplicons were cleaned using MinElute® Gel Extraction Kit (QIAGEN) according to the manufacturer’s instructions. Amplicon DNA concentrations were determined using the Quant-iT PicoGreen dsDNA reagent and kit (Invitrogen). Using the amplicon pool, we carried out pyrosequencing using primer A and Titanium chemistry on a 454 Life Sciences Genome Sequencer FLX instrument (Roche) at the DNA Sequencing Facility at the Kunming Institute of Zoology, Chinese Academy of Sciences.