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Genomic analysis of human noroviruses using combined Illumina–Nanopore data

Whole-genome sequence analysis of noroviruses is routinely performed by employing a metagenomic approach. While this method- ology has several advantages, such as allowing for the examination of co-infection, it has some limitations, such as the requirement of high viral load to achieve full-length or near full-length genomic sequences. In this study, we used a pre-amplification step to obtain full-length genomic amplicons from 39 Canadian GII isolates, followed by deep sequencing on Illumina and Oxford Nanopore platforms. This approach significantly reduced the required viral titre to obtain full-genome coverage. Herein, we compared the cov- erage and sequences obtained by both platforms and provided an in-depth genomic analysis of the obtained sequences, including the presence of single-nucleotide variants and recombination events.


Introduction
Noroviruses are the most common agents causing acute gastroenteritis, leading to an estimated 684 million illnesses and approximately $60 billion in societal costs worldwide (Bartsch et al., 2016). There are currently no vaccines or therapeutics licensed against norovirus (Cates et al., 2020). Norovirus transmission primarily occurs by person-to-person contact through the faecal-oral route or contaminated food and surfaces (Teunis et al., 2015).
Noroviruses belong to the Caliciviridae family and have a 7.5kb, positive-sense, single-stranded RNA genome that is enclosed in a non-enveloped icosahedral capsid (Green 2013). The genome is organized into three open reading frames (ORFs). ORF1 encodes a polyprotein that is cleaved into six non-structural viral proteins, including the RNA-dependent RNA polymerase (RdRp). ORF2 encodes VP1, the major structural capsid protein, and ORF3 encodes VP2, a minor structural capsid protein (Green 2013). Noroviruses are considered fast-evolving viruses (de Graaf, van Beek, and Koopmans 2016), and their genomes are tremendously diverse due to the accumulation of point mutations and recombination (Parra 2019). To date, noroviruses are classified into at least ten genogroups. Noroviruses are further classified into at least 49 genotypes based on the diversity of ORF2 and 60 types based on the diversity of the RdRP gene (Chhabra et al., 2019).
Whole-genome sequence (WGS) analysis of noroviruses, which is often carried out through metagenomic approaches, has allowed for source attribution, lineage analysis, identification of recombination events, and variant analysis (Parra and Green 2015;Nasheri et al., 2017;Petronella et al., 2018). However, a high viral titre is required to obtain a full-genome coverage through metagenomics approaches (Nasheri et al., 2017). Alternatively, amplicon-based sequencing using sequence-specific primer sets decreases the viral load requirement in the sample but introduces amplification bias and assumes conserved viral synteny, which could lead to overlooking genomic variations (Cotten et al., 2014). Parra and colleagues have recently developed a method to amplify full-length norovirus GII genomes, followed by the use of the Illumina platform to obtain deep sequencing data on the full-genome amplicons (Parra et al., 2017).
Third-generation sequencing devices such as Oxford Nanopore's MinION, which can produce long reads up to 100s of kilobases, have become the method of choice to elucidate viral recombination and to identify subgenomic sequences (Viehweger et al., 2019). On the other hand, second-generation sequencing technologies like Illumina, despite a low error rate, are restricted by a read length of ≤300 nt, which, even using pairedend strategies, can limit contiguous assembly when there are repeat or multicopy sequences exceeding the read length. This results in shorter contigs and can considerably complicate the investigation of recombination and the identification of subgenomic sequences. However, the adoption of MinION sequencing for routine surveillance of viruses has been limited due to concerns of sequence accuracy (Bull et al., 2020). To overcome this concern, we performed amplicon-based long-read MinION and short-read paired-end Illumina MiSeq sequencing on matched norovirus-positive stool samples.
The aim of this study is to combine Illumina and Oxford Nanopore sequencing data to reconstruct a highly accurate consensus sequence of the norovirus isolates and to provide insight into the viral recombination and genetic diversity.

Samples
The norovirus GII-positive samples are from the British Columbia Centre for Disease Control (BMH19-089 to BMH19-137), obtained in winter and spring 2019, and from the archive of the National Food Virology Reference Centre at Health Canada (BMH11-021, BMH12-030, BMH 13-039, BMH14-054, BMH14-056, BMH15-059, BMH15-063, BMH16-074, BMH16-077, BMH18-086, and BMH18-087), which were obtained from winter 2011 to spring 2018. The presence of norovirus GII RNA was confirmed by droplet digital PCR (Bio-Rad, Hercules, California, USA) using the probes and primers that were described previously (Nasheri et al., 2017;Petronella et al., 2018). The samples either were from an outbreak or were sporadic. An outbreak includes more than two epidemiologically linked cases with more than one norovirus-positive sample. This study has been granted an exemption from requiring ethics approval by Health Canada, and formal consent was not required because the study participants were anonymized.

RNA extraction and full-length amplicon generation
Full-length amplicons were generated as described before (Parra et al., 2017). Briefly, 10 per cent stool suspensions were clarified by centrifugation (6000 × g for 5 min), and the supernatant was filtered through a 0.45-µM, and then a 0.22-µM filter (Millipore, Etobicoke, Ontario, Canada). RNA was extracted from the filtrate using the MagMax Viral RNA Isolation Kit (Ambion) following the manufacturer's recommendations. Complementary DNA (cDNA) was synthesized from the viral RNA using the Tx30SXN primer (GACTAGTTCTAGATCGCGAGCGGCCGCC-CTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (Katayama et al., 2002) and the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Amplification of the full-length genome was performed using a set of primers that target the conserved regions of the 5 ′ -and 3 ′ -end of GII noroviruses (GII1-35: GTGAATGAAGATGGCGTCTAACGACGCTTCCGCTG and Tx30SXN) and the SequalPrep Long PCR Kit (Invitrogen) following manufacturer's recommendations.

Viral RNA load determination
Viral titres were determined by droplet digital PCR (Bio-Rad, Hercules, California, USA) using the probes and primers that were described previously (Nasheri et al., 2017;Petronella et al., 2018).

Sanger sequencing
Samples that did not generate full-length replicons after three attempts were subjected to dual typing by Sanger sequencing. For this purpose, we performed conventional (reverse transcription polymerase chain reaction) RT-PCR targeting of a 570-bp product that includes the 3 ′ -end of the polymerase region and the 5 ′ -end of the major capsid gene followed by Sanger sequencing, as described previously (Cannon et al., 2019).

Illumina sequencing
Norovirus libraries were constructed using the NexteraXT DNA Library Preparation Kit and Nextera XT Index Kit v2 according to the manufacturer's instructions (Illumina Inc.). Paired-end Illumina sequencing was performed on a MiSeq instrument (v3 chemistry, 2 × 300 bp) according to manufacturer instructions (Illumina Inc.).

Oxford Nanopore sequencing
Norovirus cDNA samples were treated with 0.16 mg/ml RNase A (100 mg/ml, Qiagen) for 30 minutes at 37 • C. Samples were subsequently size selected using modified Solid Phase Reversible Immobilization (SPRI) magnetic beads to remove DNA fragments <1,500 bp (Hosomichi et al., 2014). Briefly, 1 ml of Ampure XP beads (Beckman Coulter) was applied to a magnetic stand and the supernatant was discarded. The beads were resuspended in 0.5 ml of 20 per cent Polyethylene glycol (PEG) and 2.5 M NaCl solution. The modified SPRI beads were added to DNA samples at a 0.35× volume and incubated for 5 minutes at room temperature. Samples were applied to a magnetic stand, the supernatant discarded, and beads washed 2× with 125 µl 80 per cent ethanol. The beads were air dried for 30 seconds, followed by incubation for 2 minutes at room temperature in 45 µl H 2 O. Samples were applied to a magnetic stand, and the eluted DNA was quantified using a Qubit 4 Fluorometer (Fisher).
DNA libraries were constructed using the Ligation Sequencing Kit 1D (SQK-LSK108) and the PCR Barcoding Expansion 1-12 Kit (EXP-PBC001) according to the manufacturer protocol (Oxford Nanopore Technologies, Oxford Science Park, UK). Twelve barcoded libraries were pooled per run and sequenced on a 1D Min-ION device (R9.4, FLO-MIN106) using FLO-MIN106 flow cells for up to 24 hours. Signal processing, base calling, demultiplexing and adapter trimming were performed using the Guppy (Guppy GPU v 3.3.3 + fa743ab).

Data availability
The complete de novo genome sequences of the 39 norovirus isolates used in this study have been uploaded to the GenBank under accession numbers: MW661246 to MW661284. All SRAs are available in the GenBank under BioProject ID PRJNA713985.

Amplicon production
To examine whether full-length amplicons could be generated for a variety of GII samples, we employed the primers that encompass 5 ′ UTR to 3 ′ UT on 57 GII-positive samples (44 were isolated in 2019, and the remaining 13 were archived samples isolated since 2011). Full-length amplicons were obtained from 39 samples, and multiple efforts for the remaining 18 samples failed to generate any full-length amplicons. Thus, we conducted conventional RT-PCR to obtain partial amplicons for dual typing of these samples by Sanger sequencing. Four out of 18 samples failed to generate any RT-PCR product for dual typing. The full-length amplicons obtained from all 39 samples were subjected to both Illumina and Nanopore sequencing as depicted in Fig. 1. The data obtained from both approaches were used to assemble the full genomes and for further sequence analysis.

Analysis of the assay sensitivity
To determine the lowest viral genome copy number that would produce full-length amplicons, we made serial dilutions for four representative samples: BMH16-77, BMH18-86, BMH19-95, and BMH19-96. The full-length amplicons generated from the highest dilution (lowest viral load) were subjected to Illumina sequencing to ensure that the full-genome sequence could still be obtained. As shown in Table 1, the lowest viral RNA titre that generated fulllength sequence ranged from 1.7 to 3.4 × 10 2 RNA copy number.

Comparison between Illumina and MinION coverage
To compare sequencing coverage along the full length of the norovirus genomes, coverage depth analysis was demonstrated for all samples (Fig. 3). For each sample, the Illumina and Nanopore reads were mapped to their corresponding consensus sequence and the sequencing depth at each nucleotide position was determined. As shown in Fig. 3 Overall, the Illumina data typically lacked coverage at both the 5 ′ and 3 ′ ends of the norovirus genomes (Fig. 3), resulting in incomplete genome assemblies and only partial coding sequence data for ORF1 (data not shown). In contrast, the Nanopore reads yielded full-length sequences, which covered both the 5 ′ and 3 ′ ends of the norovirus genome. Additionally, using the Nanopore reads made it possible to obtain complete and/or partially complete sequences of the 5 ′ and 3 ′ UTR regions, with the exception Figure 1. Schematic representation of experimental approach for norovirus de novo whole-genome sequencing, assembly, and bioinformatics analysis. Norovirus viral RNA was extracted from stool samples, followed by full-length cDNA amplification. cDNA was sequenced on an Illumina MiSeq platform and an Oxford Nanopore MinION device to obtain high-quality short-read and long-read data, respectively. Illumina and Nanopore reads were processed to remove adaptor and barcode sequences, followed by the quality trimming of Illumina reads and length filtering of Nanopore reads. A combined approach using both Illumina and Nanopore reads was used to produce de novo full-length norovirus genomes. Genome annotation and downstream analyses were performed using the norovirus assemblies.
of the first 35 nt at the 5 ′ end, which are from the primer used for amplification.

Phylogenetic analysis
Phylogenetic analysis of the 39 Canadian de novo assembled norovirus strains from this study was performed for ORF1, ORF2, and ORF3 ( Supplementary Fig. S1A-C), as well as the full genomes (Fig. 4). The epidemiologically linked samples such as BMH-19-108 to BMH-19-115 and BMH-19-127 to BMH-19-132 cluster closely together across all three ORFs, providing genetic evidence to the epidemiological data. As expected for norovirus infections, interhost variations were not observed between the linked samples.

Variant analysis
The structural domains of the capsid include an N-terminus, a highly conserved shell, and two protruding spike domains (P1 and hypervariable P2) (Debbink et al., 2012;Smith and Smith 2019). The capsid protein is a major target for the host immune system. Consequently, certain changes within the antigenic determinants of the capsid protein could lead to antigenic diversification and host immune evasion (Lindesmith et al., 2013;Kendra et al., 2021). To investigate potential capsid protein variations in the norovirus samples, SNV analysis was conducted for each strain using highly similar GenBank references ( Fig. 5 and Supplementary Table S3). From Fig. 5, the majority of the SNVs resulting in amino acid changes were observed in the P2 domain. This trend was observed for all of the genotypes in this study. For GII.1, the linked outbreak samples BMH19-108, BMH19-109, BMH19-110, BMH19-111, BMH19-112, and BMH19-113 all have changes in the P2 domain at T325M, H374Q, and N293S relative to the reference strain (GenBank accession MK753033). While N293S seems to be present in many GII isolates including AHV83708 and AGI96399, we could not find any evidence for T325M and H374Q in the Gen-Bank database. Interestingly, I453V in the P1 domain was only observed in BMH19-112 and BMH19-113 and not in the rest of the linked samples. BMH19-117 and BMH19-125, samples from genotype GII.2, showed changes in the N-terminus (S24N), P1 (V256I), P2 (V319I, V335I, and V373I), and P1 (V440I) (GenBank accession KJ407074). The majority of samples (17/39) in this study belong to the GII.4[P16] genotype (BMH16-074, BMH18-085, BMH19-090, BMH19-092, BMH19-093, BMH19-094, BMH19-097, BMH19-127, BMH19-128, BMH19-129, BMH19-132, and BMH19-137) and, with the exception of BMH19-137, contain a common V377A change in the P2 domain relative to the reference strain (GenBank accession MK762635). This biochemically conserved change has been reported in many isolates in the GenBank including AGS08159 and ATI15266. The norovirus GII.6 strains BMH19-118, BMH19-119, and BMH19-145 all have an S174P change in the conserved shell domain, as well as a G354Q variation in the P2 domain of BMH19-118 and BMH19-119 (GenBank accession MT731279). These changes have been reported in many isolates including AHI59154 and ADR30514.
The norovirus RdRp (NS7) is the core enzyme for RNA replication. Its structure is highly similar to those of other positive-strand RNA viruses and can be described as a partially closed right hand, with fingers, thumb, and palm subdomains (Deval et al., 2017). The fingers and palm domains interact to form a channel. Within this channel, there are seven conserved motifs named A-G, which interact with the template, the nascent RNA, and the NTPs for RNA synthesis and comprise the active site (Deval et al., 2017). As shown in Fig. 6, the majority of the SNVs resulting in amino acid changes in RdRP were observed in the fingers and the palm domains. Also, as previously mentioned, GII.P16 was the dominant polymerase type, associated with three capsid genotypes: GII.4, GII.2, and GII.1. Similar to what has been observed for the VP1 protein, the linked outbreak samples BMH19-108, BMH19-109, BMH19-110, BMH19-111, BMH19-112, and BMH19-113 all have the same changes in the fingers and palm domains at T1294S, S1401T, and V1446A relative to the reference strain (GenBank accession MK753033) (Supplementary Table S4). The K1646R substitution is one of the few amino acid changes in the thumb domain that was observed in the majority of the GII.4[P16] isolates, while the S1401T change in the palm domain was observed for most GII.P16 isolates regardless of the capsid genotype ( Fig. 6 and Supplementary Table S4).

Discussion
The approach implemented by Parra and colleagues for full-length amplicon generation of GII samples has the potential to revolutionize the genome analysis of noroviruses. This method has recently been employed to obtain full-genomic sequences from Figure 3. Coverage profiles of norovirus samples using Illumina and Nanopore read data. Reads were mapped to the de novo consensus sequence for each genotype and read type. Depth across the genome at each nucleotide position for Illumina (blue) and Nanopore (black) is shown.
hundreds of archival samples since the 1970s (Tohma et al., 2021). Herein, we applied a modified version of this approach on 57 norovirus GII-positive samples and obtained full-length amplicons for 39 samples (>67 per cent). We performed Sanger sequencing to determine the genotype of the remaining samples. The failure of these samples to generate full amplicons could be explained by the presence of RT-PCR inhibitors as the viral titre for these samples are above the limit of the assay. For future applications, diluting the samples could potentially reduce the effect of RT-PCR inhibitors and aid in obtaining full-length amplicons (Nasheri et al., 2020). We have also determined the lowest viral RNA load that would render full-genomic amplicons for four representative samples and demonstrated that 170-346 genome copies would be enough for WGS using this technique. This is promising as the level of natural contamination for certain highrisk foods, such as oysters, is within or even higher than this range (Le Guyader et al., 2009). Therefore, this approach has the potential to be employed for WGS analysis of naturally contaminated food products in the absence of an enrichment strategy (Nasheri, Vester, and Petronella 2019).
The difference between the approach used for sequencing in this study and the method employed by Parra and colleagues is that we did not perform amplicon purification for size selection; nevertheless, we obtained full-genomic sequences for all the fulllength amplicons that were subjected to Illumina and Nanopore sequencing. Amplicon purification is often laborious, especially if applied to a large number of samples. Herein, we demonstrated that this step could be skipped without sacrificing the quality of the obtained sequences.
Coverage analysis of norovirus genomes demonstrated the utility of combining long-read Nanopore and short-read Illumina sequencing data to obtain full-length de novo norovirus genomes. Using an approach that consisted only of Illumina sequencing, we often obtained incomplete genome assemblies. Indeed, decreased sequencing coverage at the 5 ′ and 3 ′ ends of the norovirus sequence is observed for the Illumina data (Fig. 3), resulting in partial assembly for ORF1 and, less frequently, ORF3. By using a combined genome assembly approach, we were able to obtain highly accurate complete norovirus genomic sequences by first extracting full-length Nanopore sequence reads followed by error correction using a combination of both long-and shortread sequencing data. Differences in coverage depth along the genomes that are observed in some of the samples (Fig. 3) are likely the result of incomplete cDNA synthesis (partial genome length amplicons) or the presence of subgenomic sequences.
In recent years, the emergence and spread of RdRp/capsid recombinant noroviruses have been reported around the world.  Table S1). It was also interesting to detect the GII.17 [P17] strain that caused several outbreaks in multiple countries during (van Beek et al., 2018Matsushima et al., 2019) from a sample that was isolated in 2015 (Supplementary Table S1). Even though GII.P16 has recently re-emerged as the dominant polymerase type, the oldest isolate in this study, BMH11-021, also has P16, but it does not cluster with the recent isolates; instead, it shows identity to older P16 isolates from 2011 (Fig. 4A). On the other hand, more recent GII.4 isolates from 2019 still show high identity to the older isolates from 2013, 2014, and 2015.
This observation, together with the fact that GII.4 has continued to be the dominant genotype for more than three decades, indicates that despite some sequence plasticity, the GII.4 capsid is evolutionarily conserved due to some fitness advantages. The phylogenetic analyses in this study further confirm that epidemiologically linked isolates, such as BMH19-108 to BMH19-115 resulting from the GII.1[P16] outbreak, are highly similar across all three ORFs (Fig. 4) and, with the exception of I453V in the P1 domain of the VP1 protein, inter-host variation was not observed (Nasheri et al., 2017;Brown et al., 2019;Zhang et al., 2020).
The P2 subdomain of the VP1 protein interacts with potential norovirus carbohydrate receptors and contains seven antigenic epitopes, A-G (Debbink et al., 2012;Lindesmith et al., 2013;Tohma et al., 2019); therefore, it is not surprising to see that the majority of the non-synonymous SNVs are concentrated in this subdomain (Fig. 5) (Cannon et al., 2017), which is absent in GII.4 Sydney[P31] isolates. In addition, BMH19-090 contains the M333V change in the epitope C of the P2 subdomain that was reported in GII.4 Sydney[P16] isolates in multiple studies (Cannon et al., 2017;Ruis et al., 2020). It has been suggested that these substitutions influence viral fitness by altering antigenicity and increasing transmissibility, receptor binding, or particle stability (Koromyslova and Hansman 2015;Lindesmith et al., 2019;Ruis et al., 2020;Kendra et al., 2021). Outbreak samples BMH19-127 to BMH19-132 have an S470T within the P1 subdomain that has not been reported before. GII.2[P16] strains have been reported in the last decade, and the S24N and V335I substitutions observed in the isolates in this study have been detected before (Tohma et al., 2017). However, V256I, V319I, V373I, and V440I substitutions were only observed in the samples from this study (Supplementary Table S3), and they are considered biochemically conserved substitutions.
The re-emerging P16 isolated in this study was associated with three capsid genotypes: GII.4, GII.2, and GII.1 (Fig. 6). The outbreak-associated GII.P16 (GII.1) isolates all have the same three substitutions T1294S, S1401T, and V1446A (Supplementary Table  S4). However, the S1401T substitution in the palm domain was also observed in GII.P16 (GII.2) and GII.P16 (GII.4) isolates. Three out of four sporadic P31 isolates in this study, which are associated with the GII.4 Sydney capsid, had R1236K substitution in the fingers domain. The GII.P12 isolates all have the V1521I substitution, and all the GII.P7 isolates have the I1519V substitution within the palm domain; however, none of these substitutions falls within the conserved motifs or the active sites, and they are all considered biochemically conserved substitutions; thus, their significance is not known.
While variant analysis using long-read data was limited to three out of thirty-nine norovirus samples (Supplementary Table  S5), putative variants were identified for BMH19-129 (ORF1 E1360R and ORF3 R153Q) and BMH13-039 (ORF2 P31L) (Supplementary Table S5). We did not find any precedent for ORF1 R1360 (reference coordinates MK762635) in the GenBank. However, precedents exist for ORF3, Q153, and ORF2 L31. Long-read data sets for norovirus variant calling may be improved in future by removing other amplicons (norovirus subgenomic sequences and contaminating amplicons) and/or increased Nanopore read depths.
In conclusion, using full-length amplicons is an efficient and sensitive method for norovirus WGS analysis. Also, our results are consistent with other reports regarding the predominance of GII.4[P16] Sydney replacing the previous GII.4[P31] Sydney, indicating the enhanced fitness of the GII.4 Sydney capsid. Continued norovirus genomic surveillance will help in the understanding of norovirus evolutionary mechanisms and the identification of emerging variants, which ultimately aid in designing future norovirus vaccines and antivirals.

Supplementary data
Supplementary data is available at Virus Evolution online.