Genomic mini ax bacteria kit

For bacterial strains that belonged to S. fonticola and E. coli species, which were the most abundant, genomic DNA was isolated with Genomic Mini AX Bacteria Kit (A&A Biotechnology, Gdynia, Poland). Then, the DNA concentration was measured (Epoch Microplate Spectrophotometer; BioTek Instruments, Agilent, Santa Clara, CA, USA) and the genomic DNA was analyzed with repetitive-sequence-based rep-PCR with ERIC primers [87 ]. After performing PCR reactions, 5 µL of the PCR products was resolved in 0.8% agarose gels (0.5xTBE) at 50 V for 2.5 h. After obtaining a band pattern for each bacterial strain, they were analyzed with GelJ [88 (link)]. Similarity trees were composed with the unweighted pair group method with arithmetic mean (UPGMA) with band difference 1.0. The similarity cut-off value for the pair of strains treated as having the same profile was set to >0.5. E. coli strains C49 and S. marcescens C19 were used for comparative purposes.

Whole bacterial DNA from six clinical extraintestinal E. coli was isolated from Luria Broth overnight cultures with use of silica column-based Genomic Mini AX Bacteria kit (A&A Biotechnology). Purified bacterial DNA was sequenced using both short- and long-read methodologies (Illumina and Oxford Nanopore Technology).
In the first step of molecular analysis, Nextera XT library preparation kit and Nextera XT Indexes (Illumina) were used for previously quantified bacterial DNA, which was simultaneously fragmented and tagged with sequencing adapters in a single-tube enzymatic reaction. Quality and quantity of libraries were assessed by fluorometry (Qubit, Thermo Fisher Scientific) and chip electrophoresis (2100 Bioanalyzer, Agilent). FASTQ reads were generated with the use of MiSeq Reagent Kit v3 (600 cycles) and MiSeq analyzer (Illumina).
In the next step, DNA libraries were prepared with the use of a Ligation Sequencing Kit (SQK-LSK109) with Native Barcoding Expansion (EXP-NBD104). Quality and quantity of libraries were assessed by fluorometry (Qubit, Thermo Fisher Scientific) and chip electrophoresis (2100 Bioanalyzer, Agilent). FASTQ reads were generated with the use of Spot-ON Flow Cell (FLO-MIN106D R9 Version) and MinION Mk1b analyzer (Oxford Nanopore Technology).

The Genomic Mini AX Bacteria kit (A&A Biotechnology, Gdynia, Poland) with gravity-operated DNA purification columns was used for the extraction of nucleic acids according to the protocol provided by the manufacturer of the kit, which allowed for the extraction of high-quality nucleic acids. DNA concentration was tested using a nanodrop device (Thermo Fisher, Waltham, MA, USA) following the manufacturer’s instructions. After verifying the degree of DNA contamination with proteins and chemical compounds, the degree of DNA fragmentation was checked by performing electrophoresis on a 2% agarose gel (90 V, 70 min). The appearance of a dense band indicated the lack of fragmentation of deoxyribonucleic acid, while fragmented DNA appeared in the form of a characteristic streak consisting of molecules of various sizes. The extracted nucleic acids were used both in multiplex PCR and in next-generation sequencing (NGS).

DNA was isolated from 1 mL of liquid cultures and from several colonies obtained from agar plates. The DNA was extracted with a Genomic Mini AX Bacteria kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s instructions. The amount of DNA used in the PCR reaction varied between 1 and 25 ng. The extracted DNA was frozen at −20 °C or directly subjected to PCR analysis.