title: Microbiome of plasmid mutant treated soil at later stage
design: To generate bacterial libraries, the first round of PCR was performed to amplify the V3-V4 region of the 16S rRNA gene using universal primers 341F and 805R which contains Nextera consensus and adaptor sequences at the forward and reverse. The PCR products were cleaned-up and amplified with primers containing Illumina dual indices and sequencing adapters using the forward index i5 and reverse i7. The PCR conditions were as follows: initial denaturation at 94 C for 3min, followed by 25 cycles of denaturation at 94 C for 30s, annealing at 55 C for 30s, extension at 72 C for 30s, and final extension at 72 C for 5min. Purified amplicon libraries were pooled and sequenced at ChunLab Inc. (South Korea) with an Illumina MiSeq system using the MiSeq Reagent Kit v2 (Illumina Inc., USA).