Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts.

Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified viral load as a critical factor in variant identification. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.

Perfect Match Genomic Landscape strategy: Refinement and customization of reference genomes.

When addressing a genomic question, having a reliable and adequate reference genome is of utmost importance. This drives the necessity to refine and customize reference genomes (RGs). Our laboratory has recently developed a strategy, the Perfect Match Genomic Landscape (PMGL), to detect variation between genomes [K. Palacios-Flores et al.Genetics 208, 1631-1641 (2018)]. The PMGL is precise and sensitive and, in contrast to most currently used algorithms, is nonstatistical in nature. Here we demonstrate the power of PMGL to refine and customize RGs. As a proof-of-concept, we refined different versions of the Saccharomyces cerevisiae RG. We applied the automatic PMGL pipeline to refine the genomes of microorganisms belonging to the three domains of life: the archaea Methanococcus maripaludis and Pyrococcus furiosus; the bacteria Escherichia coli, Staphylococcus aureus, and Bacillus subtilis; and the eukarya Schizosaccharomyces pombe, Aspergillus oryzae, and several strains of Saccharomyces paradoxus. We analyzed the reference genome of the virus SARS-CoV-2 and previously published viral genomes from patients' samples with COVID-19. We performed a mutation-accumulation experiment in E. coli and show that the PMGL strategy can detect specific mutations generated at any desired step of the whole procedure. We propose that PMGL can be used as a final step for the refinement and customization of any haploid genome, independently of the strategies and algorithms used in its assembly.

Trends of mutation accumulation across global SARS-CoV-2 genomes: Implications for the evolution of the novel coronavirus.

To understand SARS-CoV-2 microevolution, this study explored the genome-wide frequency, gene-wise distribution, and molecular nature of all point-mutations detected across its 71,703 RNA-genomes deposited in GISAID till 21 August 2020. Globally, nsp1/nsp2 and orf7a/orf3a were the most mutation-ridden non-structural and structural genes respectively. Phylogeny of 4618 spatiotemporally-representative genomes revealed that entities belonging to the early lineages are mostly spread over Asian countries, including India, whereas the recently-derived lineages are more globally distributed. Of the total 20,163 instances of polymorphism detected across global genomes, 12,594 and 7569 involved transitions and transversions, predominated by cytidine-to-uridine and guanosine-to-uridine conversions, respectively. Positive selection of nonsynonymous mutations (dN/dS >1) in most of the structural, but not the non-structural, genes indicated that SARS-CoV-2 has already harmonized its replication/transcription machineries with the host metabolism, while it is still redefining virulence/transmissibility strategies at the molecular level. Mechanistic bases and evolutionary/pathogenicity-related implications are discussed for the predominant mutation-types.