Viral na sv kit

After bacterial culture, colonies were resuspended in 500 μL of water and bacterial pellets were digested using MagNA Pure 96 DNA Bacterial Lysis Buffer and proteinase K. DNA extraction was performed on a MagNA Pure 96 System (Roche Diagnostics, Penzberg, Germany) using the MagNA Pure 96 DNA and Viral NA SV Kit. Quantification and purity checks (260/280 and 260/230 ratios) were performed using NanoDrop (Thermo Scientific, Waltham, MA, United States) before external sequencing by Helixio (Saint-Beauzire, France³). Qubit quantification was carried out prior to sequencing. Library preparations were made using 1 ng of DNA and the Nextera XT DNA Library Preparation Kit (Illumina, Inc., San Diego, CA, United States) and validation of the libraries was performed on a bioanalyzer with the High Sensitivity DNA Assay kit (Agilent, Santa Clara, CA, United States) in order to obtain sizes ranging from 250 to 1,500 base pairs (bp). Paired-end sequencing was then performed on a NextSeq500 (Illumina). Quality was controlled using FastQC v0.11.3 (Wingett and Andrews, 2018 (link)). De novo assemblies were produced using SPAdes v3.10.1 (Bankevich et al., 2012 (link)).

Initially, 30 multiresistant A. butzleri isolates were selected to perform NGS and genomic resistance marker identification. Bacterial DNA was extracted using the MagNA Pure 6 DNA and Viral NA SV Kit, and purification was performed from bacterial lysis on a MagNA Pure 96 System (Roche Applied Science, Manheim, Germany). Spectrophotometry using NanoDrop Technologies (Wilmington, DE, USA) was performed on all DNA samples for quantification and purity checks (260/280 and 260/230 ratios). Following DNA extraction, NGS was performed using an Illumina HiSeq 4000 machine (Integragen, Evry, France), quality tests were run using FastQC v0.11.9 (47 (link)), and raw (.fastq) data were cleaned using Sickle v1.33 (48 ) and assembled using SPAdes v3.10.1 (49 (link)). Species identification of all isolates was also performed using FastANI v1.1 (50 ) against A. butzleri reference genomes NCTC 12481 (51 (link)) and RM4018 (29 (link)). The studied genomes are available in the NCBI database under BioProject PRJNA798874, and the corresponding identifiers are presented in Table 2. Finally, the determination of associated antimicrobial genomic resistance markers was performed using Prokka v1.14.6 (52 (link)) annotation software and the Comprehensive Antibiotic Resistance Database (CARD) Resistance Gene Identifier webtool (card.mcmaster.ca/analyze/rgi) (53 (link)).

FeLV proviral DNA was determined in all samples by extracting total nucleic acids (TNA) [25 (link)] from 100 µL of EDTA anticoagulated whole blood using the MagNA Pure 96 instrument (Roche Diagnostics AG, Rotkreuz, Switzerland) and the Viral NA SV Kit (Roche Diagnostics AG, Rotkreuz, Switzerland), following the protocol as described before [17 (link)]. The proviral DNA copy number was quantified by real-time quantitative polymerase chain reaction (qPCR) as described previously [26 (link)]. To verify the quality and quantity of the TNA a qPCR was performed for the detection of feline albumin on all 934 TNA samples as previously described [27 (link)]. In a previous study, it was shown that FeLV provirus quantitative real-time PCR showed a high analytical sensitivity (detection of 1 copy/PCR) and a high analytical specificity (detection of all three FeLV subtypes, no false-positive results in SPF cats) [28 (link)].