16s rDNA
the genespecific sequences used in this protocol target the 16S rRNA gene V4 variable region. PCR reaction volume were containing 10pmol of both primers (5-GTGCCAGCMGCCGCGGTAA*-3[515F] and barcoded 5-GGACTACHVGGGTWTCTAAT*-3 [806R]), 5L Accuprime buffer II, 0.2L Accuprime Hifi polymerase (Thermo Fisher Scientific, Waltham, WA, USA), H2O(WMB) and 2 L DNA to be a total volume of 50 l