design: The V3-V4 hyper-variable regions of the 16S rRNA gene were amplified with two rounds of PCR. The concentration of the purified amplicons was measured using Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the quality of a set of amplicons was checked using DNA 7500 chips onto a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). All libraries were pooled at equimolar concentration in order to generate equivalent number of raw reads with each library. The final pool had a diluted concentration of 5 nM to 20 nM and was used for sequencing. Amplicon libraries were mixed with 15% PhiX control according to the Illuminas protocol. For this study, one-sequencing run was performed using NovaSeq 6000