Bacterial 16S rRNA gene for pyrosequencing was amplified by
PCR using a 10-nucleotide barcoded forward primer
50-AGAGTTTGATCCTGGCTCAG-30 and the reverse primer
50-TTACCGCGGCTGCTGGCAC-30 , which target at the hypervariable
V1–V3 region (Lu et al., 2012b, 2014). The amplicons were purified
and quantified then were mixed and sequenced on a Roche 454
GS-FLX+ according to standard protocols.