High Throughput SARS-CoV-2 Genome Sequencing from 384 Respiratory Samples Using the Illumina COVIDSeq Protocol.

The emergence of the Coronavirus Disease 2019 (COVID-19) pandemic has fostered the use of high-throughput techniques to sequence the entire severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and track its evolution. The present study proposes a rapid and relatively less expensive sequencing protocol for 384 samples by adapting the use of an Illumina NovaSeq library to an Illumina MiSeq flow cell instrument. The SARS-CoV-2 genome sequences obtained with Illumina NovaSeq and those obtained using MiSeq instruments were compared with the objective to validate the new, modified protocol. A total of 356 (94.6%) samples yielded interpretable sequences using the modified Illumina COVIDSeq protocol, with an average coverage of 91.6%. By comparison, 357 (94.9%) samples yielded interpretable sequences with the standard COVIDSeq protocol, with an average coverage of 95.6%. Our modified COVIDSeq protocol could save 14,155 euros per run and yield results from 384 samples in 53.5 h, compared to four times 55.5 h with the standard Illumina MiSeq protocol. The modified COVIDSeq protocol thus provides high quality results comparable to those obtained with the standard COVIDSeq protocol, four times faster, while saving money.

First cases of infection with the 21L/BA.2 Omicron variant in Marseille, France.

The SARS-CoV-2 21K/BA.1, 21L/BA.2, and BA.3 Omicron variants have recently emerged worldwide. To date, the 21L/BA.2 Omicron variant has remained very minority globally but became predominant in Denmark instead of the 21K/BA.1 variant. Here, we describe the first cases diagnosed with this variant in south-eastern France. We identified 13 cases using variant-specific qPCR and next-generation sequencing between 28/11/2021 and 31/01/2022, the first two cases being diagnosed in travelers returning from Tanzania. Overall, viral genomes displayed a mean (±standard deviation) number of 65.9 ± 2.5 (range, 61-69) nucleotide substitutions and 31.0 ± 8.3 (27-50) nucleotide deletions, resulting in 49.6 ± 2.2 (45-52) amino acid substitutions (including 28 in the spike protein) and 12.4 ± 1.1 (12-15) amino acid deletions. Phylogeny showed the distribution in three different clusters of these genomes, which were most closely related to genomes from England and South Africa, from Singapore and Nepal, or from France and Denmark. Structural predictions highlighted a significant enlargement and flattening of the surface of the 21L/BA.2 N-terminal domain of the spike protein compared to that of the 21K/BA.1 Omicron variant, which may facilitate initial viral interactions with lipid rafts. Close surveillance is needed at global, country, and center scales to monitor the incidence and clinical outcome of the 21L/BA.2 Omicron variant.

SARS-CoV-2 quasi-species analysis from patients with persistent nasopharyngeal shedding.

At the time of a new and unprecedented viral pandemic, many questions are being asked about the genomic evolution of SARS-CoV-2 and the emergence of different variants, leading to therapeutic and immune evasion and survival of this genetically highly labile RNA virus. The nasopharyngeal persistence of infectious virus beyond 17 days proves its constant interaction with the human immune system and increases the intra-individual mutational possibilities. We performed a prospective high-throughput sequencing study (ARTIC Nanopore) of SARS-CoV-2 from so-called "persistent" patients, comparing them with a non-persistent population, and analyzing the quasi-species present in a single sample at time t. Global intra-individual variability in persistent patients was found to be higher than in controls (mean 5.3%, Standard deviation 0.9 versus 4.6% SD 0.3, respectively, p < 0.001). In the detailed analysis, we found a greater difference between persistent and non-persistent patients with non-severe COVID 19, and between the two groups infected with clade 20A. Furthermore, we found minority N501Y and P681H mutation clouds in all patients, with no significant differences found both groups. The question of the SARS-CoV-2 viral variants' genesis remains to be further investigated, with the need to prevent new viral propagations and their consequences, and quasi-species analysis could be an important key to watch out.

Simultaneous SARS-CoV-2 Genome Sequencing of 384 Samples on an Illumina MiSeq Instrument through Protocol Optimization.

In the present study, we propose a high-throughput sequencing protocol using aNextera XT Library DNA kit on an Illumina MiSeq instrument. We made major modifications to this library preparation in order to multiplex 384 samples in a single Illumina flow cell. To validate our protocol, we compared the sequences obtained with the modified Illumina protocol to those obtained with the GridION Nanopore protocol. For the modified Illumina protocol, our results showed that 94.9% (357/376) of the sequences were interpretable, with a viral genome coverage between 50.5% and 99.9% and an average depth of 421×. For the GridION Nanopore protocol, 94.6% (356/376) of the sequences were interpretable, with a viral genome coverage between 7.0% and 98.6% and an average depth of 2123×. The modified Illumina protocol allows for gaining EUR 4744 and returning results of 384 samples in 53.5 h versus four times 55.5 h with the standard Illumina protocol. Our modified MiSeq protocol yields similar genome sequence data as the GridION Nanopore protocol and has the advantage of being able to handle four times more samples simultaneously and hence is much less expensive.

Evolution of SARS-CoV-2 Strains in Senegal: From a Wild Wuhan Strain to the Omicron Variant

The coronavirus disease 2019 (COVID-19) is a contagious disease caused by a new coronavirus called SARS-CoV-2. The first case was discovered in Wuhan, China, in December 2019, raising concerns about the emergence of a new coronavirus that poses a significant public health risk. The objective of this study, based on data collected and sequenced at the Institut de Recherche en Santé, de Surveillance Epidémiologique et de Formations (IRESSEF), is to characterize the pandemic evolution, establish a relationship between the different strains in each wave, and finally determine the phylodynamic evolution of the pandemic, utilizing microreact simulations. The study shows that SARS-CoV-2 strains have evolved over time and the variability of the virus is characterized by sequencing during each wave, as is its contagiousness (the speed at which it spreads). The pandemic has spread at a rate of 44.34 cases/week during the first wave. Twelve weeks later it has risen to 185.33 cases/week during the second wave. Twenty-three weeks into the pandemic, the numbers have reached 681.77 cases/week during the third wave. During the fourth wave, the rate of infection was found to decrease slightly at 646 cases/week between early December 2021 and mid-January 2022. Data collected during this study also provided us with a geographical distribution of COVID-19, indicating that the epidemic started in Dakar before spreading inland.

Heterogeneity of the Omicron variant in Senegal.

•Omicron variant continues to progress in Senegal with the appearance of new contaminations.•IRESSEF detected the first positive case of the Omicron variant on Friday, December 3, 2021.•Since this date, the number of Omicron variant infections has increased over the weeks.•Molecular surveillance of the Omicron variant allowed us to identify a strong variation of this variant in our country.

Analysis of SARS-CoV-2 Variants From 24,181 Patients Exemplifies the Role of Globalization and Zoonosis in Pandemics.

After the end of the first epidemic episode of SARS-CoV-2 infections, as cases began to rise again during the summer of 2020, we at IHU Méditerranée Infection in Marseille, France, intensified the genomic surveillance of SARS-CoV-2, and described the first viral variants. In this study, we compared the incidence curves of SARS-CoV-2-associated deaths in different countries and reported the classification of SARS-CoV-2 variants detected in our institute, as well as the kinetics and sources of the infections. We used mortality collected from a COVID-19 data repository for 221 countries. Viral variants were defined based on ≥5 hallmark mutations along the whole genome shared by ≥30 genomes. SARS-CoV-2 genotype was determined for 24,181 patients using next-generation genome and gene sequencing (in 47 and 11% of cases, respectively) or variant-specific qPCR (in 42% of cases). Sixteen variants were identified by analyzing viral genomes from 9,788 SARS-CoV-2-diagnosed patients. Our data show that since the first SARS-CoV-2 epidemic episode in Marseille, importation through travel from abroad was documented for seven of the new variants. In addition, for the B.1.160 variant of Pangolin classification (a.k.a. Marseille-4), we suspect transmission from farm minks. In conclusion, we observed that the successive epidemic peaks of SARS-CoV-2 infections are not linked to rebounds of viral genotypes that are already present but to newly introduced variants. We thus suggest that border control is the best mean of combating this type of introduction, and that intensive control of mink farms is also necessary to prevent the emergence of new variants generated in this animal reservoir.

Dynamics of Variants of Concern (VOC) of SARS-CoV-2 during the Different Waves of COVID-19 in Senegal

Background: In Senegal, the incidence of SARS-CoV-2 evolved with four successive epidemic waves. The first wave started in March 2020 with low virus variability, whilst the second outbreak, which started in December 2020, was dominated by the Alpha variant. The third wave took place in June 2021, and the fourth at the end of November 2021. Our interest was to investigate the involvement of variants of concern during these four waves and to track the viral diversity of SARS-CoV-2. Methodology: During the four waves of the pandemic, 276,876 nasopharyngeal swabs were analyzed at the Institut de Recherche en Santé, de Surveillance Epidémiologique et de Formation (IRESSEF). Of these, 22,558 samples tested positive for SARS-CoV-2 by RT-PCR. Then, the virus genomes were sequenced in 817 positive samples using the ARTIC Network of Oxford Nanopore Technologies (ONT). In addition, 10% of the negative samples in RT-PCR new variants were also targeted for the detection of new and previously undescribed variants. Results: Our data have overall shown that the Senegalese strains are very similar to each other or closely related to other strains, such as Gambia, France etc. During the first wave, the most common clade found was 19A (67.5%) and a majority of the samples were of the B.1 (50%) lineage. We noted more diversity during the second wave where clade 20A (38.4%) was more frequent, followed by clade 20B (31.52%) and 20I (9.74%). At the level of lineages, we identified variants of concern as B.1.1.7 (9.74%) and B.1.617.2 (0.86%). In the third wave, we observed at the clade level with mainly 21A (32.63%) and 21J (16.84%). During the fourth wave at the end of November 2021, we mainly identified clade 21K Omicron variant 21K (B.1.1.529 and BA.1) (80.47%) and Delta variant (21A, 21J, and 21I) (AY.103, AY.122, AY.122.1, AY.26, AY.34, AY.36, AY.4, AY.48, AY.57, AY.61, and AY.87) (14.06%). Impact: SARS-CoV-2 diversity may affect the virus's properties, such as how it spreads, disease severity, or the performance of vaccines, tools, or other public health and social measures. Therefore, such tracking of SARS-CoV-2 variants is not only of public interest, but also highlights the role some African institutes such as IRESSEF with surveillance capabilities through the real-time sequencing of SARS-CoV-2 genomes in the local context. Conclusion: In Senegal, the SARS-CoV-2 pandemic has disrupted the organization of the health system. IRESSEF contributed to put in place strategies to respond effectively to the expectations of medical authorities by providing them with data on the strains circulating in Senegal at each moment of the epidemic.

Occurrence of a substitution or deletion of SARS-CoV-2 spike amino acid 677 in various lineages in Marseille, France.

Great concerns have been raised about SARS-CoV-2 variants over the past six months. At the end of 2020, an increasing incidence of spike substitutions Q677H/P was described in the USA, which involved six independent lineages. We searched for changes to this amino acid in the sequence database of SARS-CoV-2 genomes obtained at the IHU Méditerranée Infection (Marseille, France) from 3634 patients sampled between February 2020 and April 2021. In seven genomes (0.2%), we found a deletion of five amino acids at spike positions 675-679 (QTQTN) including Q677, and in 76 genomes (2.3%) we found a Q677H substitution. The 83 genomes were classified in ten different Pangolin lineages. Genomes with a spike Q677 deletion were obtained from respiratory samples collected in six cases between 28 March 2020 and 12 October 2020 and in one case on 1 February 2021. The Q677H substitution was found in genomes all obtained from respiratory samples collected from 19 January 2021 and were classified in seven different lineages. Most of these genomes (41 cases) were of UK variant. Two others were classified in the B.1.160 Pangolin lineage (Marseille-4 variant) which was first detected in July 2020 in our institute but was devoid of this substitution until 19 January 2021. Also, eight genomes were classified in the A.27/Marseille-501 lineage which was first detected in our institute in January 2021 and which either harboured or did not harbour the Q677H substitution. Thus, the spike Q677H substitution should be considered as another example of convergent evolution, as it is the case of spike substitutions L18F, E484K, L452R, and N501Y which also independently appeared in various lineages.