Genomic tip 100 g column

Total DNA was purified from overnight culture using Qiagen Genomic-tip 100/G columns (Qiagen, Germantown, MD, United States) per the manufacturer’s instructions. Whole-genome sequencing (WGS) was performed on the Illumina HiSeq 2500 platform and the Oxford MinIon Nanopore platform. FastQC (version 0.11.9)¹ and NanoQC (Version 0.9.4)² were used to assess the quality of short reads generated by Illumina and long reads generated by MinIon, respectively. High-quality long reads were assembled de novo using Canu (version 2.1.1)³ (Koren et al., 2017 (link)). Contigs were circularized by Circlator⁴ using the following parameters: merge_min_id, 85; merge_breaklen, 1,000; bwa_opts, -x ont2d; assembler, canu (Hunt et al., 2015 (link)). High-quality short reads were used to correct circularized contigs with two iterations of Pilon (version 1.24)⁵ (Walker et al., 2014 (link)) correction and one round of Racon (version 1.4.3)⁶ (Vaser et al., 2017 (link)) polishing. All programs were run with default parameters unless otherwise specified.

Genomic DNA was extracted using Genomic-tip 100/G columns (Qiagen, Hilden, Germany). All isolates were sequenced with >30-fold coverage on an Illumina MiSeq instrument using Nextera XT libraries, 50 samples dual-indexes multiplexing and 2 × 300 cycles v3 reagent cartridges. Paired-end reads were assembled de novo using SPAdes 3.9.0^{75 (link)} with default parameters. In addition, selected isolates were resequenced on a PacBio RSII or Sequel instrument in order to generate completely closed reference sequences. Genomes were annotated using Prokka 1.7^{76 (link)} with the H. pylori species database and ncRNAs were identified using Infernal^{77 (link)} and Rfam 11.0^{78 (link)}. Annotations of OMPs were manually curated using the 26695 and J99 reference strains as well as multiple sequence alignment and phylogenetic comparisons. For each patient, reference genomes and raw read data were submitted to NCBI under BioProject PRJNA490474. GFF Annotations files were deposited on FigShare doi:10.6084/m9.figshare.7188239.

Genomic DNA was obtained from bacteria grown at 37°C on Difco^TM Cystin Heart Agar (BD247100, France), or chocolate blood agar medium with PolyVitex (PVX, Biomerieux, Marcy l’Etoile, France). DNA was extracted and purified using QIAGEN^® Genomic-tip 100/G columns and QIAGEN^® Genomic DNA Buffer sets from suspension calibrated to 3 McFarland units according to the manufacturer’s recommendations. Purified DNA was quantified in a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Rodano, MI, Italy) using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific), and the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), following the manufacturer’s instructions.

Genomic DNAs were obtained from vegetative cells grown at 37°C on 5% horse blood agar plates. DNA was purified using the QIAGEN® Genomic-tip 100/G columns and QIAGEN® Genomic DNA Buffer Set. Briefly, bacterial colonies were harvested by scraping the agar surfaces from 16 to 18 h-old Petri dishes. Cell pellet was resuspended in 3.3 ml of Buffer B1 containing 7 μl of RNase A (100 mg/ml, QIAGEN) and incubated at 37°C for 45 min with 350 μl of lysozyme (100 mg/ml, Roche) and 100 μl of proteinase K stock solution (QIAGEN). Following addition of 1.2 ml of Buffer B2, DNA lysate was further incubated at 50°C for 1 h. Particle-free sample was next applied to the equilibrated QIAGEN Genomic-tip and DNA purification on anion-exchange resin processed according to the manufacturer’s recommendations. After isopropanol precipitation, genomic DNA was resuspended in 400 μl of 10 mM Tris HCl (pH 8) for at least 2 h at 50°C.
DNA solutions were transferred to a 0.22 μm sterile Ultrafree-MC spin filter (Millipore) and centrifuged for 2 min at a maximal speed of 8000 × g to ensure the complete removal of live forms of B. anthracis from DNA. Viability testing was systematically performed before DNA was taken out of the BSL-3 facility. An aliquot of each DNA preparation (a quarter) was spread on Petri dishes and grown at 37°C for 24 h.

High molecular weight DNA was extracted from L. monocytogenes cultures using Qiagen Genomic-tip 100/G columns and a modified manufacturer’s protocol as previously described [41 (link)] with the addition of mutanolysin with the proteinase K step followed by incubation at 50 °C for 1 h. Ten micrograms of DNA were sheared to a targeted size of 20 kb using a g-TUBE (Corvaris, Woburn, MA) and concentrated using 0.45X volume of AMPure PB magnetic beads (Pacific Biosciences, Menlo Park, CA) following the manufacturer’s protocol. Sequencing libraries were created using 5 μg of sheared, concentrated DNA and the PacBio DNA Template Prep Kit 2.0 (3Kb - 10Kb) according to the manufacturer’s protocol. The library was bound with polymerase P5 followed by sequencing on a Pacific BioSciences (PacBio) RS II sequencing platform with chemistry C3 and the 120 min data collection protocol.

H. somni isolates were previously sequenced using SMRT sequencing as previously described^{21 (link)} for consensus genome assembly and identification of SSRs. For the present study, the USDA-ARS-USMARC-63250 strain was revived from a −80 °C glycerol stock by streaking onto Chocolate II agar plates (Becton Dickinson Co. Sparks, MD) in 5% CO₂ at 37 C for 24–48 h. An individual, well-separated colony was used to inoculate a 10 ml culture of brain heart infusion (BHI) broth supplemented with 2X Veterinary Fastidious Medium (Thermo Fisher Scientific, Waltham, MA)(Thermo Scientific, Waltham, MA) in a 50 ml conical tube, which was incubated at 37 °C with shaking at ~190 rpm for 24 h. Cells were pelleted and genomic DNA extracted using the Qiagen DNeasy Blood & Tissue kit and Genomic-tip 100/G columns according to the manufacturer’s protocol. Genomic DNA quantity and quality were assessed by spectrophotometry using a NanoDrop (Thermo Fisher, Waltham, MA).