Nuclisens easymag

Ribonucleic acid (RNA) extraction and PCR testing were performed at the Sunnybrook Research Institute in Toronto, Ontario. All swab, tissue and guano samples were stored at -80°C prior to testing. For oral, rectal or nasal swab samples, RNA extractions were performed using 140 µL of sample via the QIAmp viral RNA mini kit (Qiagen, Mississauga, Ontario) or the Nuclisens EasyMag using Generic Protocol 2.0.1 (bioMérieux Canada Inc., St-Laurent, Québec) according to manufacturer’s instructions. The RNA from guano samples (80 mg) were extracted via the QIAmp viral RNA mini kit and eluted in 40 µL in containment level 3 at the University of Toronto. Tissue samples were thawed, weighed, minced with a scalpel, and homogenized in 600 µL of lysis buffer using the Next Advance Bullet Blender (Next Advance, Troy, New York, US) and a 5 mm stainless steel bead at 5 m/s for 3 minutes. The RNA from 30 mg tissue samples was extracted via the RNeasy Plus Mini kit (Qiagen, Mississauga, Ontario) or the Nuclisens EasyMag using Specific Protocol B 2.0.1; RNA was eluted in 50 µL. All extractions were performed with a positive and negative control. Extraction efficiency between kits was assessed through comparison of positive extraction controls.

Nucleic acid extraction and sequencing was carried out using a modified version of a tiling amplicon-based method to enrich the culture supernatant for viral RNA, which we previously used to sequence mumps virus from a 2010 Ontario outbreak^{13 (link)}. We extracted RNA using either the QIAamp Viral RNA Mini Kit (Qiagen, Mississauga, ON) or the NucliSENS easyMAG instrument. For the initial eight samples we performed amplification of 18 overlapping amplicons, of mean length 977 bp. We optimised the protocol to reduce the number of amplicons, so for the last 19 samples we sequenced 9 amplicons of mean length 1958 bp (Supplementary Dataset 2 for primers). Amplification of the fragments in 96 well plates was performed on a SimpliAmp thermal cycler using the superscript III One Step RT-PCR system (Invitrogen,Thermo Fisher Scientific).
Amplicon fragments from individual samples were pooled together in equal amounts and cDNA concentration checked using a Qubit fluorometer. Mumps cDNA libraries were prepared with the Nextera XT kit. We checked the quality of the indexed libraries by Bioanalyzer. Sequencing on the Illumina MiSeq instrument was performed with V2 reagent kit (2 × 150 bp, Illumina Inc. San Diego, California, USA), according to the manufacturer’s instructions.