Singular bacteria colonies were picked from CSM agar (pH 6.5) and incubated in 20 mL Tryptic Soy Broth medium in 150 mL baffled shaking flasks at 120 rpm and 28°C overnight. HMW gDNA was extracted according to the instructions of the Quick‐DNA HMW MagBead Kit (Zymo Research).
To assess the quantity and purity of the obtained DNA, 260/280 nm absorption ratios and concentrations were measured with a photometer (Nano Photometer NP80; IMPLEN) and a Qubit 4 fluorometer with the Qubit 1X dsDNA HS Assay‐Kit (Thermo Fisher Scientific). To confirm the high molarity of the gDNA, fragment sizes were analyzed with a Femto Pulse capillary electrophoresis instrument (Agilent Technologies).
When samples passed the quality control, shearing of 8 µg gDNA in 150 µL Elution Buffer was conducted with g‐TUBEs (Covaris), utilizing 1700g in a tabletop centrifuge. This yielded DNA fragments with a size of ca. 12 kbp, as confirmed with Femto Pulse. Subsequently, HiFi libraries were prepared according to the SMRTbell prep kit 3.0 manual, fusing barcoded adapters to the samples (Pacific Biosciences). Libraries were stored at −20°C until the day of sequencing, where primers and the polymerase bound the samples with the Sequel II Binding Kit 3.2 (Pacific Biosciences), closely following the manufacturer's recommendations.
To assess the quantity and purity of the obtained DNA, 260/280 nm absorption ratios and concentrations were measured with a photometer (Nano Photometer NP80; IMPLEN) and a Qubit 4 fluorometer with the Qubit 1X dsDNA HS Assay‐Kit (Thermo Fisher Scientific). To confirm the high molarity of the gDNA, fragment sizes were analyzed with a Femto Pulse capillary electrophoresis instrument (Agilent Technologies).
When samples passed the quality control, shearing of 8 µg gDNA in 150 µL Elution Buffer was conducted with g‐TUBEs (Covaris), utilizing 1700g in a tabletop centrifuge. This yielded DNA fragments with a size of ca. 12 kbp, as confirmed with Femto Pulse. Subsequently, HiFi libraries were prepared according to the SMRTbell prep kit 3.0 manual, fusing barcoded adapters to the samples (Pacific Biosciences). Libraries were stored at −20°C until the day of sequencing, where primers and the polymerase bound the samples with the Sequel II Binding Kit 3.2 (Pacific Biosciences), closely following the manufacturer's recommendations.