Genomic tip 20 kit

Cells of the A. altamirensis strain C2P003 were cultured in R2 broth for two days at 25 °C and washed two times with 0.3% NaCl. Genomic DNA was extracted by the Genomic-Tip 20 Kit (Qiagen, Hilden, Germany) as described previously [19 (link)]. The fragment size, quantity and quality of DNA were assessed on a 1% agarose gel and with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). DNA was sequenced using the Pacific Biosciences (PacBio) RS II sequencing platform at Eurofins Genomics (Konstanz, Germany). Sequence reads were de novo assembled using the PacBio hierarchical genome assembly process (HGAP4). The assembling resulted in one contig with an average genome coverage of 124×. The genome sequence was circulated with the Circlator v. 1.5.5 [91 (link)].

Cells were grown in 500 mL of W medium at 55°C for 4 d under H₂/CO₂ (80/20 [v/v]), recovered by centrifugation (12,000 rpm) at 4°C for 15 min, and washed twice with dH₂O. Cell pellets were suspended in TE buffer. The suspension was then subjected 5 times to a freeze-thaw treatment of freezing at −80°C for 30 min and thawing at 55°C for 10 min. The treated solution was incubated overnight at 55°C following the addition of SDS, proteinase K, NaCl, and RNase A, at final concentrations of 0.5% (w/v), 100 μg mL⁻¹, 10 mM, and 10 μg mL⁻¹, respectively. After the incubation, the solution was gently mixed by inversion with double the volume of TE-saturated phenol. A half-volume of chloroform was added, and the solution was kept on ice for 5 min before being centrifuged at 14,000 rpm for 15 min at 4°C. Sodium acetate (pH 8) was then added to the transferred upper aqueous layer to a final concentration of 0.3 M. The solution was gently mixed with an equal volume of isopropanol and centrifuged at 14,000 rpm for 15 min at 4°C. The precipitate was washed with an equal volume of 70% (v/v) ethanol and centrifuged at 14,000 rpm for 15 min at 4°C. The resultant precipitate was dried and resuspended in 100 μL of TE buffer. The sample of genomic DNA was further purified using a Genomic-tip 20 kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions.

Stenotrophomonas sp. 169 was cultured in R2A medium for 2 days at 25∘C. The cells were washed three times with 0.3% NaCl to remove exopolysaccharides, and genomic DNA was subsequently extracted by the Genomic-Tip 20 Kit (Qiagen) according to the manufacturer’s instructions. The purity, fragment size, and quantity of the isolated DNA were assessed on a 1% agarose gel and by using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). DNA was sequenced using the Pacific Biosciences (PacBio) RS II sequencing platform at GATC Biotech AG (Konstanz, Germany). From the genomic DNA, a 20 kb insert size library was prepared and sequenced using 2 SMRT cells and a 240 min movie time. Sequence reads were de novo assembled using the PacBio hierarchical genome assembly process (HGAP3). The assembly resulted in one contig with an average genome coverage of 195x. The genome sequence was circularized with Circlator ver. 1.5.5 and deposited in the GenBank database under accession no. CP061204.