Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.

COVID-19 is a complex disease phenotype where the underlying microbiome could influence morbidity and mortality. Amplicon and metagenomic MinION based sequencing was used to rapidly (within 8 hours) identify SARS-CoV-2 and assess the microbiome in nasopharyngeal swabs obtained from patients with COVID-19 by the ISARIC 4C consortium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

Background The mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure. Results Investigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323. Conclusions These data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.