16s of gut
The 16S rDNA target region of the ribosomal RNA gene were amplified by PCR 95 C for 5 min, followed by 30 cycles at 95 C for 1 min , 60 C for 1 min , and 72 C for 1 min and a final extension at 72 C for 7 min using primers listed in the table. PCR reactions were performed in triplicate 50 L mixture containing 10 L of 5  Q5 Reaction Buffer , 10 L of 5  Q5 High GC Enhancer 1.5 L of 2.5 mM dNTPs, 1.5 L of each primer ( 10 M), 0.2 L of Q5 High Fidelity DNA Polymerase , and 50 ng of temp late DNA. Related PCR reagent s were from New England Biolabs USA.Amplicons were extracted from 2% agarosegels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer's instructions and quantified using ABI StepOnePlus Real Time PCR System (Life Technologies Foster City, USA) USA). Purified amplicons were pooled in equimolar and paired end sequenced ( PE250) on an Illumina platform according to the standard protocols.