MiSeqOSV125 P08F08
Genomic DNA (gDNA) was extracted using Qiagen DNeasy Blood and Tissue Kit protocol in 96-well plates. Dual-indexed primers for multiplexed amplicon sequencing of the V3-V4 region of the 16S rRNA gene were designed. PCR was performed using the normalized gDNA as template and Phusion High-Fidelity DNA Polymerase for 25 cycles with 0.05 M of each primer. Samples were pooled by plate, purified using the DNA Clean & Concentrator-5 kit and eluted in water, quantified by NanoDrop, and combined in equal proportions into a library. The library was quantified using Qubit 1 HS Assay, diluted to 4.2 nM, and loaded at 10 pM onto Illumina MiSeq platform for 300-bp paired-end sequencing using MiSeq Reagent Kit v2 (500-cycle), or loaded at 21 pM using MiSeq Reagent Kit v3 (600-cycle) depending on the desired sequencing reads.