Bigdye terminator kit version 3

Using the method described by Marmur [32 (link)], the genomic DNA from strain F20-122^T was extracted and purified for 16S rRNA and rpoB´ gene analysis and for genome sequencing. The quality of the DNA was checked by (1%) agarose gel electrophoresis. DNA quantification was determined by spectrophotometry (DeNovix DS-11 FX, DeNovix Technologies, Wilmington, Delaware, USA) and fluorometry (Qubit 3.0 Fluorometer, Thermofisher Scientific, USA). The 16S rRNA and rpoB´ genes were amplified by PCR [33 ] with the universal primers ArchF and ArchR [34 (link),35 (link)] and the primers designed by Fullmer et al. [36 (link)], respectively. The PCR products were sequenced by StabVida (Oeiras, Portugal) using the Sanger method and the same primers. For the 16S rRNA gene sequencing, primers 16RB36 (GGA CTA CCA GGG TAT CTA) and 16RD34 (GGT CTC GCT CGT TGC CTG) were also used. Sequencing reactions were carried out using a BigDye terminator kit version 3.1 from Applied Biosystems. A draft genome sequence of strain F20-122^T was also determined in this study using a whole-genome shotgun strategy. After the DNA quality control, a library was constructed using the Kappa HyperPrep library preparation kit. The generated DNA libraries were sequenced in the lllumina Hiseq 4000 platform, using 150 bp paired-end sequencing reads (StabVida, Oeiras, Portugal).

Viral RNA was extracted from the patients’ serum (200 μL stored at −80 °C) using NucleoMag 96 Virus (Macherey-Nagel, Düren, Germany) and automated KingFisher™ Magnetic Particle Processors (Thermo Fisher Scientific Inc., Waltham, MA, USA) in accordance with the manufacturer’s instructions. Serum samples from healthy subjects were used as negative controls. The RNA was eluted in 50 μL of nuclease-free distilled water, and reverse transcribed using the SuperScript III reverse transcriptase protocol (Thermo Fisher Scientific, Waltham, Massachusetts, USA): the cDNA was amplified by means of nested polymerase chain reaction (PCR) using GoTaq^® DNA Polymerase (Promega, Madison, WI, USA). The primers for the first and second rounds of NS5B amplification and the PCR conditions have been previously described [42 (link),43 (link)].
The fragments obtained by means of PCR were purified using a commercial purification kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany), and then sequenced bi-directionally using a BigDye Terminator Kit version 3.1 (Applied Biosystems, CA, USA) in accordance with the manufacturer’s instructions.
The sequencing products were purified from a 10 μL sample by means of precipitation in an ethanol/sodium acetate mixture. Finally, the sequences were determined using an automated DNA sequencer (ABI PRISM 3130 XL Genetic Analyser, Applied Biosystems).

DNA was isolated using DNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Polymerase chain reaction (PCR) was performed using KAPATaq Extra HotStart Ready Mix with dye (NIPPON Genetics, Tokyo, Japan). Serogroups [35 (link), 36 (link)] and phylogenetic groups [37 (link)] were determined by PCR. Multilocus sequence typing (MLST) was determined according to Tartof et al. [38 (link)]. Genes of fosA, fosA3, fosC2 [22 (link)], fosB2, fosC, fosX [26 (link)], fosB [21 (link)], fosA3/4 [25 (link)], and fosKP96 [24 (link)] were detected by PCR using the primers described previously. Gene of fosA5 was detected by PCR using primer set: 5′-ACTGAATCACCTGACCCTGG-3′ and 5′-CGCATAATGGGTGTAGTCGC-3′. Full nucleotide sequences of six genes (murA, uhpT, glpT, uhpA, ptsI, and cyaA) were determined by a combination of direct sequencing and primer walking with the respective PCR products. PCR primer sequences are given in Table 1. The sequencing was performed with Big Dye Terminator Kit version 3.1 and 3730xI DNA analyzer (Applied Biosystems, Carlsbad, CA) at Hokkaido System Science (Sapporo, Japan).