Genomic DNA was isolated from the collected samples using a Nexttec 1-Step DNA Isolation Kit (nexttec Biotechnologie GmbH, Leverkusen, Germany). PCR used universal primers (27Fmod and 338R) for 16S rRNA gene sequencing, as previously described36 (link),37 (link). PCR used 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, Japan). Cycling conditions were: 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, CA, USA) and quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies Japan). After quantification, mixed samples were prepared by pooling approximately equal amounts of each amplified DNA. Samples were sequenced using a MiSeq Reagent Kit V3 (300 × 2 cycles) and a MiSeq sequencer (Illumina, CA, USA), following the manufacturer's instructions.
Thermal cycling was performed in a Veriti Thermal Cycler (Life Technologies Japan, Tokyo, Japan). Cycling conditions were: 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, CA, USA) and quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies Japan). After quantification, mixed samples were prepared by pooling approximately equal amounts of each amplified DNA. Samples were sequenced using a MiSeq Reagent Kit V3 (300 × 2 cycles) and a MiSeq sequencer (Illumina, CA, USA), following the manufacturer's instructions.
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