Whole genome sequencing
To fully understand the genetic relatedness of RV1 derived strains to the original RV1 strain, complete genome sequencing was performed on 20 RV1 derived strains and 24 wild type G1P[8] strains selected on the basis of specimen availability and a high viral load (i.e., Ct values<30). Rotavirus RNA extraction, library preparation and sequencing were performed according to an unpublished rotavirus whole-genome sequencing protocol generously provided by the US Centers for Disease Control and Prevention (CDC) with the following modifications: the use of the most recent version of New England Biolabs (NEBNext Ultra II) RNA library preparation kit for Illumina and Illumina MiSeq kit, and an updated extraction method. Briefly, 30% stool filtrates were produced by diluting 300 µL of liquid stool specimen or 300 mg of solid stool specimen in 700 µL of PBS, vortex-homogenized, and centrifuged for 15 mins at 14,000 rcf. Finally, samples were filtered through Durapore-PVDF Ultrafree-MC Centrifugal Filters with 0.65 µm pore size (EMD-Millipore, Canada). Viral dsRNA was extracted using QIAamp® Viral RNA Mini Kit (Cat No./ID: 52904, QIAGEN, USA) following manufacturer’s recommendation. The quality of purified dsRNA was assessed by rotavirus RT-qPCR. Five microliters of dsRNA in the 10-100 ng concentration range, as measured by Qubit™ RNA HS Assay Kit (Q32852, ThermoFisher Scientific, USA) was used to generate cDNA libraries with NEBNext® Ultra II RNA Library Prep Kit for Illumina® (E7770S, New England Biolabs, USA) and indexed using NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (E6440S, New England Biolabs, USA) following manufacturer’s protocols. cDNA libraries were quantified using Qubit™ dsDNA HS Assay Kit (Q32851, ThermoFisher Scientific, USA). The average size of the libraries was visualized on Agilent TapeStation D4200 with High Sensitivity DNA D1000 ScreenTape analysis kit (both from Agilent, USA). Rotavirus RT-qPCR was used again to detect rotavirus insert in the cDNA libraries. Selected libraries were quantified using NEBNext® Library Quantification kit for Illumina® (E7630S, New England Biolabs, USA) on ABI 7500 real-time PCR instrument (ThermoFisher, USA). Libraries were normalized to 4 nM concentration and sequenced on Illumina MiSeq platform at the Bacterial Typing Unit of the Alberta Public Health Laboratory Edmonton (Alberta Precision Laboratories) with Illumina MiSeq Kit V3 (150-cycle, cat MS-102-3001; Illumina, USA).