Agilent high sensitivity d5000 screentape system

Genomic DNA from the 22 environmental strains tested on HeLa cells was isolated as described above. The quantity was measured using both a Qubit 2.0 fluorometer with a Qubit dsDNA HS assay kit (Invitrogen, Thermo Fisher Scientific) and a 2200 TapeStation instrument with a genomic DNA ScreenTape assay (Agilent Technologies Inc., Santa Clara, CA, USA). Genomic libraries were prepared using a Nextera XT kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol and quantified by capillary electrophoresis, applying the Agilent High Sensitivity D5000 ScreenTape system (Agilent Technologies Inc.). Libraries were sequenced on a MiSeq machine (Illumina) using v2 reagents with 2 × 250-bp paired-end reads. Consequently, 81.6% to 98.8% of bases in sequencing reads had quality scores of 30 (Q30) or higher. De novo genome assembly was performed using CLC Genomic Workbench v5 (Qiagen GmbH). Rapid Annotation using Subsystem Technology (RAST server; https://rast.nmpdr.org/) was used for functional annotation of proteins (61 (link)).

To study phage virions, we isolated the faecal VLP fraction and sequenced dsDNA phages as previously described^{19 (link)}. Briefly, the VLPs were extracted from 500 mg of faeces using high-speed centrifugation followed by filtration through a 0.45 µm membrane. Any free-DNA debris was digested prior to lysing the VLPs, whereafter the DNA was purified using a two-step phenol/chloroform extraction protocol. Finally, the DNA was purified using the DNeasy Blood&Tissue kit (Qiagen Cat#69506) according to the manufacturer’s protocol. Library preparation was done with the NEBNext Ultra II FS DNA library prep kit (New England Biolabs Cat#E7805L) and the NEBNext Multiplex Oligos for Illumina dual indexes (New England Biolabs Cat#E7600S) according to manufacturer’s instructions. Quality and concentration of the VLP libraries were assessed with the Qubit dsDNA HS kit (ThermoFisher Cat#Q32854) and with the Agilent High Sensitivity D5000 ScreenTape system (Agilent Technologies). Libraries were sequenced using 2×150 bp paired-end chemistry on an Illumina NovaSeq 6000 platform with the S4 Reagent Kit v1.5, 300 cycles (Illumina Cat#20028312) at the Core Facility Genomics of the Amsterdam UMC.

Genomic DNA was isolated using the QIAamp DNA Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s procedure with a protocol for Gram-negative bacteria. Final elution was performed with nuclease-free water. DNA quality was assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). The quantity was measured using both the Qubit 2.0 Fluorometer with Qubit dSDNA HS Assay Kit (Invitrogen, Thermo Fisher Scientific, Wilmington, USA) and the 2200 TapeStation Instrument with Genomic DNA ScreenTape Assay (Agilent Technologies Inc., St Clara, CA, USA). Libraries were prepared using the Nextera XT kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol and quantified by capillary electrophoresis applying the Agilent High Sensitivity D5000 ScreenTape System (Agilent Technologies Inc.). Libraries were sequenced on the MiSeq machine (Illumina) using v2 reagents with 2 × 250 bp paired-end reads. Consequently, 90.2 and 82.4% of bases of sequencing reads had quality scores ≥ Q30 for K1609 and K670, respectively. De novo genome assembly was performed using CLC Genomic Workbench v5 (Qiagen). Plasmid DNA was isolated using AccuPrep Plasmid Mini Extraction Kit (Bioneer Company, Daejeon, South Korea) according to the manufacturer’s procedure.