Miseq reagent kit v3 600 cycle

In the MiSeq sequencing, the eight healthy piglets and six piglets with signs of splay leg were included. Sequencing libraries for the MiSeq run were prepared using the Trio RNA-Seq Library Preparation Kit (NuGEN Technologies, San Carlos, CA, USA). The library concentration was measured using a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Paisley, UK) and an Agilent High Sensitivity DNA Kit (2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA, USA). Based on the concentration, pooling and normalization to 2 nM were performed. The library pools were diluted in RNase-free water. Paired-end sequencing was performed with a MiSeq Reagent Kit v3 600 cycles on the MiSeq instrument (Illumina, San Diego, CA, USA) at the National Veterinary Institute, Uppsala, Sweden. The quality of the dataset was assessed using the FastQC software [53 ].

The same amplicons generated on NS3, NS5A and NS5B, used for Sanger analysis, were sequenced on Illumina MiSeq NGS platform (Illumina, San Diego, CA, USA). Amplicon purification and quantification were performed by Agencourt AMPure XP (Beckman Coulter, Villepinte, France) and Qubit dsDNA Assay Kit (ThermoFisher Scientific, Waltham, MA, USA), respectively. Amplicons from the same patient were pooled and diluted to a final concertation of 0.2 ng/μl and pools were subsequently used for NGS library generation which was performed using Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA). The library generated was then diluted and sequenced with MiSeq Reagent Kit v3 (600-cycles) (Illumina, San Diego, CA, USA) on MiSeq platform. Before starting our sequencing experiment, we used the online ‘Sequencing Coverage Calculator’ (Illumina, San Diego, CA, USA) [17 ] to estimate the sequencing conditions that allow achieving a depth of coverage (100,000X), where coverage is the number of reads that align to a known reference sequence. This is an essential information because multiple observations per base are needed to obtain a reliable call and, in addition, coverage is also used as a unit for the statistical power of sequencing data [18 ].

Sample libraries were prepared using reagents and procedures according to the manufacturer’s instructions (Illumina, 2013). Previously published primers targeting the hypervariable region V3–V4 of the 16s bacterial rDNA [20 (link)], which results in a 465 bp amplicon, were used.
The library validation was performed in a Bioanalyzer 2100 (Agilent Technologies, California, USA), and quantified using the Kappa Library Quant Kit for Illumina (Illumina Inc., San Diego, CA, USA), according to the manufacturer’s instructions [21 ]. Sequencing was performed using MiSeq Reagent Kit v3 (600 cycles) (Illumina Inc., San Diego, CA, USA) in an Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA), following a previously published protocol [22 (link)].

The esophageal microbiota profile was assessed on the additional biopsies obtained during endoscopy. Biopsies were mechanically destroyed through high-speed shaking with beads using Tissue Lyzer II (Qiagen, Hilden, Germany). After, bacterial DNA was extracted with QIAamp DNA Microbiome Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA concentration was determined using the Qubit dsDNA HS Assay kit and Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Purified bacterial DNA was amplified by targeting the V3-V4 region of the bacterial 16S rRNA gene. Libraries were prepared using the QIAseq 16S region panel for the V3-V4 region (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Sequencing libraries were labeled with different multiplex indexing barcodes for each sample. The presence of the target sequences was evaluated with the Agilent TapeStation (Santa Clara, CA, USA) using the D1000 ScreenTape and Reagents. Libraries’ quantifications were assessed using the QIAseq Library Quant (Qiagen, Hilden, Germany). Amplicons were sequenced using the Illumina Miseq platform (Miseq Reagent Kit v3 600 cycles, Illumina, CA, USA). Finally, sequences were trimmed, filtered, merged, and clustered into operational taxonomic units (OTUs) using CLC Genomics Workbench and CLC Microbial Genomics Module v.21 (Qiagen, Hilden, Germany).