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The bacterial hypervariable regions V3–V4 of 16S rRNA genes were amplified using the primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′), with a unique barcode sequence at the 5′ end. Equimolar amounts of the PCR amplicons from different samples were mixed and purified.