Study area and collection of deer samples
Adult and yearling white-tailed deer were sampled between November 1 and December 31, 2021 as part of the Department of Natural Resources’ annual Chronic Wasting Disease (CWD) surveillance program Natural and Forestry (MNRF) of Ontario. Samples were collected from deer harvested by hunters in southwestern and eastern Ontario and included nasal swabs and RPLN. Samples were collected by staff wearing disposable masks and gloves. Nasal swabs were stored in individual 2-ml tubes with ~1 ml of universal transport medium (Sunnybrook Research Institute (SRI)), and RPLN tissues were stored dry in 2-ml tubes. After collection, samples were immediately chilled in ice packs and then transferred to a −20°C freezer where they were kept for up to 1 week. The samples were then transferred to a −80°C freezer where they were kept until analysis. Location, date of harvest and demographic data (age/sex) were recorded for each animal when available.
Screening and detection by PCR
RNA extractions and PCR testing of samples collected from deer were performed at the SRI in Toronto, Ontario. RNA extractions were carried out with 140 µl of nasal swab sample spiked with shielded RNA enterovirus (Asuragen; using Nuclisens EasyMag using generic protocol 2.0.1 (bioMérieux Canada) according to the manufacturer’s instructions; l RNA was eluted in 50 µl. Tissue samples were thawed, weighed, minced with a scalpel, and homogenized in 600 µl lysis buffer using the Next Advance Bullet Blender (Next Advance) and a 5 mm stainless steel bead at 5 ms−1 for 3 min RNA was extracted from 30 mg tissue samples using Specific Protocol B 2.0.1 using Nuclisens EasyMag RNA was eluted in 50 µl RT– PCR was performed using the Luna Universal Probe One-Step RT–qPCR Kit (New England BioLabs, NEB; A SARS-CoV-2 5′ UTR and Specific multiplex RT–PCR were used for RNA detection 47. Quantstudio 3 software (Thermo Fisher Scientific; was used to determine cycle threshold (Ct). All samples were run in duplicate and samples with Ct <40 for both target RT–PCR and shielded RNA enterovirus in at least one lysed representation were considered positive. For tissue samples, the presence of inhibitors was assessed using a 1:5 dilution of one of the replicates. Samples were considered inconclusive if no shielded enterovirus was detected or if only one RT-PCR target was detected and re-extracted for further analysis. Samples were considered indeterminate if they were inconclusive after reextraction or if no original material remained. Presumptive positive samples were further analyzed for human RNAse P to rule out possible human contamination9. The original material of presumptive positive samples detected in the SRI was sent to the Canadian Food Inspection Agency (CFIA) for confirmatory PCR testing. The MagMax CORE Nucleic Acid Purification Kit (Thermo Fisher Scientific) and the automated KingFisher Duo Prime Magnetic Extraction System were used to extract total RNA with shielded RNA enterovirus. Enteroviral shielded RNA was used as an exogenous extraction control. A multiplex RT-PCR specific for SARS-CoV-2 E and nucleocapsid (N) was used for confirmatory detection of RNA7. The master mix for qRT–PCR was prepared with TaqMan Fast Virus 1-step Master Mix (Thermo Fisher Scientific) according to the manufacturer's instructions. Reaction conditions were 50 °C for 5 min, 95 °C for 20 s, 40 cycles of 95 °C for 3 s, then 60 °C for 30 s. Runs were performed using a 7500 Fast Real-Time PCR System (Thermofisher, ABI). Samples with Ct <36 for both RT–PCR targets were considered positive.
WGS
WGS was performed at both SRI and CFIA using independent extraction and sequencing methods. At SRI, DNA was synthesized from extracted RNA using 4 μl of LunaScript RT SuperMix 5× (NEB) and 8 μl of nuclease-free water, and added to 8 μl of extracted RNA. Complementary DNA synthesis was performed under the following conditions: 25 °C for 2 min, 55 °C for 20 min, 95 °C for 1 min and holding at 4 °C.
The ARTIC V4 primer set (was used to generate amplicons from cDNA. Specifically, two multiplex PCR mosaic reactions were prepared by combining 2.5 μl cDNA with 12.5 μl Q5 High-Fidelity 2 × Master Mix (NEB), 6 μl of nuclease-free water. and 4 μl of 10 μM ARTIC V4 primer pool (Integrated DNA Technologies). PCR cycling was then performed as follows : 98 °C for 30 s followed by 35 cycles of 98 °C for 15 si 63 °C for 5 s min.
PCR reactions were then pooled and cleaned with 1× ratio sample purification beads (Illumina) at a 1:1 bead-to-sample ratio. Amplicons were quantified using the Qubit 4.0 fluorometer using the dsDNA 1× High Sensitivity Assay (HS) kit (Thermo Fisher Scientific) and sequencing libraries prepared with the Nextera DNA Flex Prep kit (Illumina) according to the manufacturer’s instructions. Paired-end sequencing (2 × 150 bp) was performed on a MiniSeq with a 300-cycle reagent kit (Illumina) with a negative control library without an input SARS-CoV-2 RNA extract.
WGS performed at CFIA used extracted nucleic acid quantified using the HS Qubit RNA Assay Kit on a Qubit Flex Fluorometer (Thermo Fisher Scientific). Eleven microliters or 200 ng of total RNA was subjected to DNase treatment using the enzyme ezDNase (Thermo Fisher Scientific) according to the manufacturer’s instructions. DNase-treated RNA was then used for library preparation and target sequence capture according to the ONETest Coronaviruses Plus Assay protocol (Fusion Genomics48). Enriched libraries were quantified using the Qubit 1 × dsDNA HS Assay Kit on a Qubit Flex Fluorometer (Thermo Fisher Scientific) and subsequently pooled into equimolar amounts prior to fragment analysis on the 4200 TapeStation System using the D5000 ScreenTape Assay (Agilent). The final pooled library was sequenced on an Illumina MiSeq using a V3 flow cell and a 600-cycle kit (Illumina).
Human specimens are received at the Ontario Public Health Laboratory for routine SARS-CoV-2 diagnostic testing (RT-PCR) from a variety of healthcare settings, including hospitals, clinics, and centers for the assessment of coronavirus disease 2019 (COVID -19). The human sample (ON-PHL-21-44225) was sequenced at the Public Health Ontario Laboratory using an Illumina-based ARTIC V4 protocol (similar to deer sequencing methods. Briefly, cDNA was synthesized using LunaScript reverse transcriptase (NEB). Amplicons were sequenced. generated with premixed ARTIC V4 primer sets (Integrated DNA Technologies). Amplicons from both groups were combined, purified with AMPure XP beads (Beckman Coulter), and quantified. Genomic libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina), and genomes were sequenced as paired-end reads (2 × 150 bp) on an Illumina MiSeq instrument.
Genomic analysis
Paired-end illumination reads from ARTIC V4 sequencing and Fusion Genomics were initially analyzed separately with the Nextflow nf-core/viralrecon (v2.3) workflow (refs. 49,50,51) which ran: FASTQC (v0.11.9) (ref. 52) read-level quality control, fastp (v0.20.1) (ref. 53) quality filtering and adapter trimming, Bowtie2 (v2.4.2 ) (ref. 54) mapping read to Wuhan-Hu-1 (MN908947.3) (ref. 55) Reference SARS-CoV-2, Mosdepth (v0.3.1) (ref. 56)/Samtools (v.1.12) (ref. 57) read map statistics calculation, iVar (v1.3.1) (ref. 58) ) ARTIC V4 primer clipping, variant calling and consensus generation; SnpEff (v5.0) (ref. 59)/SnpSift (v4.3t) (ref. 60) for prediction and annotation of variant effects; and Pangolin (v3.1.20) (ref. 61) with PangoLEARN (2022-01-05), Scorpio (v0.3.16) (ref. 62) and Constellations (v.0.1.1) was used for the assignment PANGO lineage63. Soft-trimmed read alignments trimmed with iVar primer were converted to fgbio ClipBam-trimmed alignments (Reads from hard-trimmed BAM files were sent to FASTQ files with “samtools fastq”. nf-core/viralrecon was returned to run in amplicon mode without iVar Primer Trim on combined Fusion Genomics and ARTIC V4 trimmed FASTQ reads to generate variant call, variant effect, and consensus sequence results used in subsequent analyses.Steps additional quality control tests to check the validity of the negative control, dropout, sample cross-contamination and excess ambiguity were performed using ncov-tools v1.8.0 (ref. 64) Mutations identified by the Nextclade ( v1.10.2) (ref. 65) with database 2022-01-05 and xlavir (v0.6.1). ) was manually searched in the ‘Lineage | Mutation Tracker’ from outbreak.info (2 February 2022) (ref. 66) for information on mutation prevalence observed globally and within Canada. Mutations were also investigated for presence in a specific lineage. includes VOCs, Michigan mink samples, and other animal samples. Finally, mutations were searched in GISAID (on February 2, 2022) to calculate the number of non-human hosts in which each mutation had been observed.
There were some limitations in genome quality and coverage that may have resulted in additional mutations not being detected. All B.1.641 samples had missing terminal domains and contained internal regions with no or low coverage when sequenced using the ARTIC amplicon scheme v4. This is a widespread problem that may explain the rarity of the 3′ proximal ORF10:L37F in GISAID. Significantly, in our samples, this meant no or <10× coverage across the five deer-derived sequences from ~27000 to 27177 (dropping ARTICv4 amplicons 90-91), which includes regions of the M gene .However, by combining ARTIC v4 sequencing with additional sequencing using probe-based enrichment, we were able to compensate for this dropout and generate high coverage and completeness (<100 positions without...