Qubit double stranded dna high sensitivity assay kit

Extracted DNA was quantified using the Qubit double-strandedDNA high-sensitivity assay kit according to the manufacturer’s instructions (Life Technologies Corp., Carlsbad, CA, USA). The Illumina libraries were prepared using the Nextera XT DNA library preparation kit and Nextera XT index primers (Illumina, san Diego, CA, USA). The library fragment size distribution was checked using the Bioanalyzer 2100 with an Agilent HS DNA kit (Agilent Technologies, Santa Clara, CA,USA) and quantified using a Qubit DNA HS assay kit in a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The generated libraries were then sequenced using a MiSeq version 2 reagent kit (Illumina) with 500 and 300 cycles. The paired-end read length of 2 X 250 bp was used for 500 cycles and 2 X 150 bp for 300 cycles on the MiSeq platform (Illumina). The quality metrics of the reads were performed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The sequences were then assembled using the A5-miseq assembler [22 (link)], and deposited into NCBI under BioProject no. PRJNA679582 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA679582). The genome sequence was annotated via the NCBI Prokaryotic Genome Annotation Pipeline [23 (link)].

Fungal isolates were grown from stock cultures on Sabouraud Dextrose Agar with antibiotics (Fort Richard Laboratories Ltd., Auckland, New Zealand) at 27–30°C. Fungal colonies were removed from agar plates and placed in sterile 1.5 mL tubes together with RNAlater (Life Technologies, Auckland, New Zealand), transferred to the laboratory, and stored at -20°C.
DNA extractions were conducted using AllPrep DNA/RNA Isolation Kits (Qiagen, Hilden, Germany). Fungal isolates, sinonasal swab pairs, and mouse fecal material were each first placed in Lysing Matrix E bead tubes (MP Biomedicals, Sydney, NSW, Australia) together with 600 μL of RLT Plus lysis buffer, and ruptured at 25 m/s for 2 × 40 s using a Tissue Lyser II (Qiagen, Hilden, Germany). Following centrifugation (5 min at 1500 g), the supernatant was transferred to DNA collection columns and processed as per manufacturer’s instructions. DNA was quantified using Qubit double-stranded DNA (high sensitivity) Assay kit (Life Technologies, Auckland, New Zealand).

Two sets of milk samples were taken from each quarter of every cow—at drying off and immediately post-calving (Fig. 1). Before milk sampling, each teat was thoroughly cleaned using a standardised protocol by experienced farm staff [14 ]. Samples were drawn from each quarter and collected in 30 ml universal tubes and placed on wet ice for transportation. DNA extractions were immediately carried out in random order on 500 μl of fresh milk using the DNeasy PowerSoil Kit (Qiagen, UK) following the manufacturer’s instructions. A DNA pool for each cow per sampling point was then created by combining an equal volume of DNA extract from each quarter, with each cow/udder being classified as the experiment unit. DNA yield and quality were assessed using a NanoDrop Spectrophotometer (ThermoFisher, UK) and a Qubit Fluorometer (ThermoFisher, UK) using the Qubit double-stranded DNA High Sensitivity Assay Kit (ThermoFisher, UK).

All ileal digesta and fecal samples obtained at postmortem were subjected to 16S rRNA gene metabarcoding targeting the V3 hypervariable region, as described previously (30 (link)). DNA concentrations of the purified libraries were then measured using a Qubit 3.0 fluorometer (Thermo Fisher Scientific, United Kingdom) using a Qubit double-stranded DNA high-sensitivity assay kit (Thermo Fisher Scientific). Using the readings obtained by the Qubit instrument, four library pools were constructed using equimolar concentrations of DNA from each sample. A reagent-only control and mock bacterial community (HM-782D; BEI Resources, ATCC, Manassas, VA) were included as part of each sequencing run to assess background contamination, sequencing error rate, and inter-run variability. On submission to the sequencing center (Edinburgh Genomics, United Kingdom), library pools were quantified using a Quant-iT PicoGreen double-stranded DNA assay kit (Thermo Fisher Scientific) to ensure a sufficient yield for sequencing. Sequencing was carried out using an Illumina MiSeq platform (Illumina, USA), using V2 chemistry and producing 250-bp paired-end reads. The generated sequences (with primers removed) are available publicly through the European Nucleotide Archive (ENA) under accession number PRJEB33396.