Amplicon
Initial PCR amplification of the whole variable region of bacterial 16S rRNA was done using the forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and the reverse primer 1492R (5-TACGGYTACCTTGTTACGACTT-3).  Each 25 L reaction volume contained 0.5 M of each primer, 1X OneTaq Hot Start Master Mix (New England Biolabs, Ipswich, MA, USA) and 20 ng DNA. PCR was performed under following cycling conditions (95 C, 5 min; 32 x [95 C, 1 min; 55 C, 1 min; 72 C, 1 min]; 72 C, 7 min;  4 C, ), and resultant amplicons checked on a 1.5% agarose gel. A second PCR amplification to cover the bacterial 16S rRNA V1-V3 hypervariable region was performed using 27F and 518R primer pairs, fused with MiSeq adapters and heterogeneity spacers compatible with Illumina indexes for multiplex sequencing