Qubit dsdna br assay kit

Both C. jejuni strains were grown overnight at 42°C on OMHA + blood plates under microaerobic conditions and genomic DNA was extracted using Epicentre Metagenomic DNA Isolation kits for Water (Illumina) according to the manufacturer’s instructions, as previously described (Clark et al., 2016 (link)). Briefly, quantification of DNA was performed by DeNovix QFX Fluorometer using Qubit dsDNA BR assay kit (Fisher Scientific). Sample libraries were prepared using a MiSeq Nextera^® XT DNA library preparation kit (Illumina). Whole genome sequencing was performed by 250 bp paired end read sequencing on the Illumina MiSeq sequencer using a MiSeq^® Reagent Kit V2 and 500 cycles on the Illumina MiSeq platform. Sequence reads were assembled into contigs using INNUca 2.6. Pangenomic annotations were performed by Roary (Supplementary Data S1). Raw reads can be accessed on NCBI under the reference number PRJNA903792. From pangenomic annotations, strain specificity, using the NCBI BLAST tool and conventional PCRs, was verified for many genes. Among these genes, two genes unique to G2008b (LpsA and DmsB) and two genes unique to D2008b (McrBC and RimP) were selected as candidates for qPCR/PCR targets for the quantification/identification of each strain in the samples (Supplementary Figure S1).

Genomic DNA was extracted from overnight MR-6/3H pure culture via the QIAamp DNA Kit (Qiagen, Hilden, Germany). The concentration of DNA was evaluated with the Qubit dsDNA BR Assay Kit on a Qubit 3.0 fluorometer (Fisher Scientific Inc., Waltham, MA, USA). Further Illumina Hiseq 1,500 (Illumina, CA, USA) paired-end (2×150) sequencing of D. tsuruhatensis MR-6/3H genome was performed by Geneanalytics LLC (Moscow, Russia). Raw read libraries were deposited at SRA (accession PRJNA903655).
Read library quality was evaluated using FastQC v0.11.9. Obtained reads were trimmed with Trimmomatic software (v0.4.8) using the automatic mode of adapter removal (Bolger et al., 2014 (link)). Unicycler v0.4.8. on the PATRIC resource center² was utilized to assemble the genome (Brettin et al., 2015 (link); Davis et al., 2020 (link)). Contigs less than 1,000 bp in length were removed from the resulting assembly. To assess the assembly quality, QUAST v5.0.2 and CheckM2 v1.0.2 tools were used (Gurevich et al., 2013 (link); Chklovski et al., 2023 (link)). Genome annotation was performed at the Rapid Annotations using the Subsystems Technology (RAST) server (Aziz et al., 2008 (link); Overbeek et al., 2014 (link); Brettin et al., 2015 (link)) and the Prokaryote Genome Annotation Pipeline. The draft genome sequence has been deposited at GenBank (accession JAPMID000000000).

Whole Genome Sequencing (WGS) using Oxford Nanopore Technology has been used for strain’s molecular identification.
6 mL of bacterial liquid culture overnight was used for DNA extraction with ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research, Irvine, CA, USA). DNA quality and quantity were determined using Nanodrop 2000 Spectrophotometer and Qubit™ dsDNA BR Assay Kit (Fisher Scientific SL, Madrid, Spain).
A sequencing library was prepared using the Rapid Barcoding Sequencing kit (SQK-RBK004; Oxford Nanopore Technologies). The barcoded sample was loaded in a MinION FLO-MIN106 v9.4.1 flow cell and was sequenced in a MinION Mk1B for 22 h approximately. The fast5 files were basecalled with Guppy 4.0.11 (Oxford Nanopore Technologies) with high accuracy basecalling mode, demultiplexed and adapters trimmed using all the parameters by default.
Taxonomy was assigned using WIMP workflow from EPI2ME platform¹⁷ . To be ensure about taxonomy classification, sequences were de novo assembled using Flye 2.8.3^{18 (link)} and the raw assembly was compared to L. plantarum strain SK151 chromosome (NZ_CP030105.1) by average nucleotide identity (ANI) using fastANI 1.32^{19 (link)}.

DNA was extracted using a ZymoBIOMICS DNA Miniprep Kit (Zymo Research). DNA quality and quantity were determined using a NanoDrop 2000 Spectrophotometer and a Qubit dsDNA BR Assay Kit (Fisher Scientific). The sequencing libraries were prepared using 200 to 400ng of DNA that were subjected to transposase fragmentation using a Rapid Barcoding Sequencing Kit (SQK-RBK004; Oxford Nanopore Technologies, ONT). Up to 12 barcoded samples were loaded in a MinION FLO-MIN106 v9.4.1 flow cell and sequenced in a MinION Mk1B or Mk1C (ONT). The fast5 files were basecalled and demultiplexed, and the adapters were trimmed using Guppy 5.0.11 (52 (link)) (ONT) (–dna_r9.4.1_450bps_sup.cfg) (–config configuration.cfg –barcode_kits SQK-RBK004 –trim_barcodes; min_score threshold default 60). Reads with a quality score of less than 10 were discarded. The run summary statistics were obtained using Nanoplot 1.38.1 (53 (link)) (–N50 –fastq).

Fat bodies were collected as described above with the exception that each sample was only reduced to 200-250 µL. 180 µL of sample was used for DNA isolation and quantification and the remainder was used to assay for protein concentration using Bradford’s reagent (VWR; M172). Protein concentration was measured using a spectrophotometer at 595 nm (VERSA max microplate reader). DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen; 69504). Isolation was performed according to the vendor’s instructions. Quantification of DNA was performed using the Qubit dsDNA BR Assay kit (Fisher Scientific; Q32850) with a Qubit 2.0 Fluorometer (Fisher Scientific; Q32866). Quantification was performed according to the vendor’s instructions. Protein concentrations were normalized to DNA concentrations.

DNA extractions from C. jejuni pellets were performed with a PowerLyser PowerSoil DNA Isolation Kit (QIAGEN, Toronto, ON, Canada) according to the manufacturer’s instructions. A FastPrep-24 5G Instrument (MP Biomedical) was used for the mechanic lysis step, consisting of two runs of 60 s at 6 m/s.
All DNA samples were quantified by DeNovix QFX Fluorometer using a Qubit dsDNA BR assay kit (Fisher Scientific) and stored at −80°C or − 20°C until further analysis.

DNA was extracted from the three cultures using the ZymoBIOMICS DNA Miniprep Kit (D4300; Zymo Research Corporation; Los Angeles, CA, USA), following the manufacturer’s recommendations. Before library preparation, extracted DNA was quantified using Qubit dsDNA BR Assay Kit (Fisher Scientific S.L; Madrid, Spain), and quality was assessed by absorbance using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific S.L; Waltham, MA, USA). The sequencing libraries were prepared using the Rapid Barcoding Sequencing Kit (SQKRBK004) from Oxford Nanopore Technologies (ONT, Oxford, UK), following the manufacturer’s recommendations. Rapid sequencing barcodes were added to the tagged ends to analyze more than one sample in a single run (barcoding). As a final step, 12 μL of each library was loaded onto a flow cell for sequencing using the Mk1c (MinION, ONT, Oxford, UK) and run for 48 h.