Genomic DNA was isolated from the collected samples using the nexttec™ 1-Step DNA Isolation Kit (nexttec Biotechnologie GmbH, Leverkusen, Germany). PCR was conducted using universal primers (27Fmod and 338R) for 16S rRNA gene sequencing, as previously described6 (link). PCR was performed using Ex Taq polymerase (Takara Bio, Shiga, Japan) and approximately 20 ng of template DNA.
Thermal cycling was performed in a Veriti Thermal Cycler (Life Technologies Japan, Tokyo) with the following cycling conditions: initial denaturation at 96 °C for 2 min, followed by 25 cycles of denaturation at 96 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min, and final extension at 72 °C. PCR amplicons were purified using AMPure XP magnetic purification beads (Beckman Coulter, Brea, CA, USA) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies Japan). After quantification, mixed samples were prepared by pooling approximately equal amounts of each amplified DNA and sequenced using MiSeq Reagent Kit V3 (300 × 2 cycles) and a MiSeq sequencer (Illumina, CA, USA), according to the manufacturer’s instructions.
Thermal cycling was performed in a Veriti Thermal Cycler (Life Technologies Japan, Tokyo) with the following cycling conditions: initial denaturation at 96 °C for 2 min, followed by 25 cycles of denaturation at 96 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min, and final extension at 72 °C. PCR amplicons were purified using AMPure XP magnetic purification beads (Beckman Coulter, Brea, CA, USA) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies Japan). After quantification, mixed samples were prepared by pooling approximately equal amounts of each amplified DNA and sequenced using MiSeq Reagent Kit V3 (300 × 2 cycles) and a MiSeq sequencer (Illumina, CA, USA), according to the manufacturer’s instructions.
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