US National Institutes of Health Prioritization of SARS-CoV-2 Variants.

Since late 2020, SARS-CoV-2 variants have regularly emerged with competitive and phenotypic differences from previously circulating strains, sometimes with the potential to escape from immunity produced by prior exposure and infection. The Early Detection group is one of the constituent groups of the US National Institutes of Health National Institute of Allergy and Infectious Diseases SARS-CoV-2 Assessment of Viral Evolution program. The group uses bioinformatic methods to monitor the emergence, spread, and potential phenotypic properties of emerging and circulating strains to identify the most relevant variants for experimental groups within the program to phenotypically characterize. Since April 2021, the group has prioritized variants monthly. Prioritization successes include rapidly identifying most major variants of SARS-CoV-2 and providing experimental groups within the National Institutes of Health program easy access to regularly updated information on the recent evolution and epidemiology of SARS-CoV-2 that can be used to guide phenotypic investigations.

Early detection of emerging SARS-CoV-2 variants of interest for experimental evaluation

Since the beginning of the COVID-19 pandemic, SARS-CoV-2 has demonstrated its ability to rapidly and continuously evolve, leading to the emergence of thousands of different sequence variants, many with distinctive phenotypic properties. Fortunately, the broad application of next generation sequencing (NGS) across the globe has produced a wealth of SARS-CoV-2 genome sequences, offering a comprehensive picture of how this virus is evolving so that accurate diagnostics, reliable therapeutics, and prophylactic vaccines against COVID-19 can be developed and maintained. The millions of SARS-CoV-2 sequences deposited into genomic sequencing databases, including GenBank, BV-BRC, and GISAID, are annotated with the dates and geographic locations of sample collection, and can be aligned to and compared with the Wuhan-Hu-1 reference genome to extract their constellation of nucleotide and amino acid substitutions. By aggregating these data into concise datasets, the spread of variants through space and time can be assessed. Variant tracking efforts have initially focused on the Spike protein due to its critical role in viral tropism and antibody neutralization. To identify emerging variants of concern as early as possible, we developed a computational pipeline to process the genomic data and assign risk scores based on both epidemiological and functional parameters. Epidemiological dynamics are used to identify variants exhibiting substantial growth over time and spread across geographical regions. Experimental data that quantify Spike protein regions targeted by adaptive immunity and critical for other virus characteristics are used to predict variants with consequential immunogenic and pathogenic impacts. The growth assessment and functional impact scores are combined to produce a Composite Score for any set of Spike substitutions detected. With this systematic method to routinely score and rank emerging variants, we have established an approach to identify threatening variants early and prioritize them for experimental evaluation.

Analysis of the ARTIC Version 3 and Version 4 SARS-CoV-2 Primers and Their Impact on the Detection of the G142D Amino Acid Substitution in the Spike Protein.

The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses. IMPORTANCE ARTIC Network primers are commonly used by laboratories worldwide to amplify and sequence SARS-CoV-2 present in clinical samples. As new variants have evolved and spread, it was found that the V3 primer set poorly amplified several key mutations. In this report, we compare the results of sequencing a matched set of samples with the V3 and V4 primer sets. We find that adoption of the ARTIC V4 primer set is critical for accurate sequencing of the SARS-CoV-2 spike region. The absence of metadata describing the primer scheme used will negatively impact the downstream use of publicly available SARS-Cov-2 sequencing reads and assembled genomes.