PCR and amplication

Total bacteria DNA was extracted from each 0.4 g musk samples by using MO BIO UltraClean fecal DNA kit (Carlsbad, CA, USA). Bacterial 16S rRNA genes in musk were PCR amplified using a set of two broadly conserved, degenerate primers targeting the V3–V4 variable regions of the 16S gene (341F: 5′–CCC TAC ACG ACG CTC TTC CGA TCT NXX XXX XXC CTA CGG GNG GCW GCA G–3′; 805R: 5′–GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA GAC TAC HVG GGT ATC TAA TCC–3′), the forward primer containing a 7-bp error-correcting barcode unique to each sample.

PCR reaction was conducted using a Mastercycler Gradient Thermal Cycler (Eppendorf) with the following condition: 94 °C for 3 min (initial dissociation), followed by 30 cycles of [94 °C for 30 s (denaturation), 60 °C for 45 s (annealing), and 72 °C for 1 min (extending)], followed by a final extension of 8 minutes at 72 °C. Negative non template controls were run for each barcoded primer pair to test for reagent contamination. Each sample was amplified and then pooled together for analysis. The PCR products were analyzed by gel electrophoresis and purified by using a PCR Purification kit (Sangon, Shanghai, China). Pyrosequencing was performed on an Illumina MiSeq 2 × 250 platform.