Recommendations for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

Kubik, Slawomir; Marques, Ana C.; Xing, Xiaobin; Silvery, Janine; Bertelli, Claire; Maio, Flavio De; Pournaras, Spyros; Burr, Tom; Duffourd, Yannis; Siemens, Helena; Alloui, Chakib; Song, Lin; Wenger, Yvan; Saitta, Alexandra; Macheret, Morgane; Smith, Ewan W.; Menu, Philippe; Brayer, Marion; Steinmetz, Lars M.; Si-Mohammed, Ali; Chuisseu, Josiane; Stevens, Richard; Constantoulakis, Pantelis; Sali, Michela; Greub, Gilbert; Tiemann, Carsten; Pelechano, Vicent; Willig, Adrian; Xu, Zhenyu

doi:10.1016/j.cmi.2021.03.029

Cited by 35 publications

(36 citation statements)

References 31 publications

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“…Rather, viral titer (Ct values) and the amount of sequencing data generated from each sample is more indicative of genome completeness than specimen type (Figure 1B,C). Our results demonstrate that samples with a Ct value at or below 30, or ~1000 SARS-CoV-2 GE/ul, with greater than 200,000 reads should produce near-complete SARS-CoV-2 genomes using using the ARTIC amplicon-based sequencing approach, in line with previously published data 11 . We observed substantial variation in Ct values from matched saliva and NP samples (Figure 2A), however the genome sequence itself was identical or nearly identical regardless of sample type (Figure 2B).…”

Section: Discussionsupporting

confidence: 90%

“…We generated high-quality genomes from saliva samples with a wide range of Ct values. A Ct value of 30 is often used as a threshold by sequencing labs, where samples with higher Ct values may not generate complete SARS-CoV-2 genomes 11 . Based on our RT-qPCR standard curve, a Ct value of 30 corresponds to ~1,000 SARS-CoV-2 genome equivalents (GE) per μL (Methods).…”

Section: Sars-cov-2 Genome Completeness Is Similar Between Np Swabs and Saliva Samplesmentioning

confidence: 99%

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Sequencing SARS-CoV-2 Genomes from Saliva

Alpert

Vogels

Breban

et al. 2021

Preprint

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Genomic sequencing is crucial to understanding the epidemiology and evolution of SARS-CoV-2. Often, genomic studies rely on remnant diagnostic material, typically nasopharyngeal swabs, as input into whole genome SARS-CoV-2 next-generation sequencing pipelines. Saliva has proven to be a safe and stable specimen for the detection of SARS-CoV-2 RNA via traditional diagnostic assays, however saliva is not commonly used for SARS-CoV-2 sequencing. Using the ARTIC Network amplicon-generation approach with sequencing on the Oxford Nanopore MinION, we demonstrate that sequencing SARS-CoV-2 from saliva produces genomes comparable to those from nasopharyngeal swabs, and that RNA extraction is necessary to generate complete genomes from saliva. In this study, we show that saliva is a useful specimen type for genomic studies of SARS-CoV-2.

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Section: Discussionsupporting

confidence: 90%

Section: Sars-cov-2 Genome Completeness Is Similar Between Np Swabs and Saliva Samplesmentioning

confidence: 99%

Sequencing SARS-CoV-2 Genomes from Saliva

Alpert

Vogels

Breban

et al. 2021

Preprint

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“…The only P.1 defining replacement not found at high frequency in our study was the deletion in ORF1ab (del 11288:11296), called in only four genomes. Deletions overlapping annealing sites of amplicon primers are associated with a strong decrease in the PCR efficiency prior to sequencing, leading to low genomic coverage [ 27 ]. Then, after applying a stringent coverage depth filter (DP > 50) for calling the genomic positions in the consensus sequences, this deleted region was flagged as low coverage.…”

Section: Resultsmentioning

confidence: 99%

Predominance of the SARS-CoV-2 Lineage P.1 and Its Sublineage P.1.2 in Patients from the Metropolitan Region of Porto Alegre, Southern Brazil in March 2021

et al. 2021

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Almost a year after the COVID-19 pandemic had begun, new lineages (B.1.1.7, B.1.351, P.1, and B.1.617.2) associated with enhanced transmissibility, immunity evasion, and mortality were identified in the United Kingdom, South Africa, and Brazil. The previous most prevalent lineages in the state of Rio Grande do Sul (RS, Southern Brazil), B.1.1.28 and B.1.1.33, were rapidly replaced by P.1 and P.2, two B.1.1.28-derived lineages harboring the E484K mutation. To perform a genomic characterization from the metropolitan region of Porto Alegre, we sequenced viral samples to: (i) identify the prevalence of SARS-CoV-2 lineages in the region, the state, and bordering countries/regions; (ii) characterize the mutation spectra; (iii) hypothesize viral dispersal routes by using phylogenetic and phylogeographic approaches. We found that 96.4% of the samples belonged to the P.1 lineage and approximately 20% of them were assigned as the novel P.1.2, a P.1-derived sublineage harboring signature substitutions recently described in other Brazilian states and foreign countries. Moreover, sequences from this study were allocated in distinct branches of the P.1 phylogeny, suggesting multiple introductions in RS and placing this state as a potential diffusion core of P.1-derived clades and the emergence of P.1.2. It is uncertain whether the emergence of P.1.2 and other P.1 clades is related to clinical or epidemiological consequences. However, the clear signs of molecular diversity from the recently introduced P.1 warrant further genomic surveillance.

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“…CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made genotypes at a fraction of at least 70% (31) and the country of origin. Clades were assigned to the genotypes using Nextclade (version 0.13.0) (32) and lineages using Pangolin (version 2.3.2) (33).…”

Section: Identification Of Emerging Mutations In Ddm Databasementioning

confidence: 99%

Mutational hotspot in the SARS-CoV-2 Spike protein N-terminal domain conferring immune escape potential

Kubik

Arrigo

Bonet

et al. 2021

Preprint

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Global efforts are being taken to monitor the evolution of SARS-CoV-2, aiming at early identification of mutations with the potential of increasing viral infectivity or virulence. We report a striking increase in the frequency of recruitment of diverse substitutions at a critical residue (W152), positioned in the N-terminal domain (NTD) of the Spike protein, observed repeatedly across independent phylogenetic and geographical contexts. We investigate the impact these mutations might have on the evasion of neutralizing antibodies. Finally, we uncover that NTD is a region exhibiting particularly high frequency of mutation recruitments, suggesting an evolutionary path on which the virus maintains optimal efficiency of ACE2 binding combined with the flexibility facilitating the immune escape.

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Recommendations for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

Cited by 35 publications

References 31 publications

Sequencing SARS-CoV-2 Genomes from Saliva

Sequencing SARS-CoV-2 Genomes from Saliva

Predominance of the SARS-CoV-2 Lineage P.1 and Its Sublineage P.1.2 in Patients from the Metropolitan Region of Porto Alegre, Southern Brazil in March 2021

Mutational hotspot in the SARS-CoV-2 Spike protein N-terminal domain conferring immune escape potential

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