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sequencing were performed according to an unpublished rotavirus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).