Qubit fluorometric quantitation qubit 4 fluorometer

Strain GD1 was cultured on NB medium overnight at 30°C. Bacterial cells were harvested and washed three times in 0.3% sterile NaCl. The extraction of ultra-pure DNA was done using the ZymoBIOMICS DNA Mini Kit (Zymo Research, USA), following the manufacturer protocol. The DNA yield was measured using Qubit Fluorometric Quantitation (Qubit 4 Fluorometer, Invitrogen^™, USA). The genome of strain GD1 was sequenced using a 2 × 300 bp paired-end run (MiSeq Reagent kit v3) on a MiSeq platform, according to manufacturer’s instructions (Illumina) in commercial service (FISABIO, Valencia, Spain). Total of 1,220,080 paired reads were generated.

Special sterile swabs were taken from all of the sampling points and protected by DNA/RNA shields (Zymo Research, Irvine, CA, USA) during transport. The extraction of ultra-pure DNA was completed using the Zymo BIOMICS DNA Mini Kit (Zymo Research) for several swabs from each sampling point, following the manufacturer’s protocol. The DNA yield was measured using Qubit Fluorometric Quantitation (Qubit 4 Fluorometer, Invitrogen™, Waltham, MA, USA). Library preparation, using Nextera XT Index Kit (FC-131-1096), and amplicon sequencing step was performed using a 2×300 bp paired-end run on a MiSeq Sequencer, according to manufacturer’s instructions (Illumina) in commercially available service (FISABIO, Valencia, Spain). To target the ITS II region, the primers [39 (link)] were as follows: forward primer ITS3_KYO2 (5′- GATGAAGAACGYAGYRAA-3′), and reverse primer ITS4_KYO1 (5′-TCCTCCGCTTWTTGWTWTGC-3′).