Multiple Mutations Associated with Emergent Variants Can Be Detected as Low-Frequency Mutations in Early SARS-CoV-2 Pandemic Clinical Samples

Genetic analysis of intra-host viral populations provides unique insight into pre-emergent mutations that may contribute to the genotype of future variants. Clinical samples positive for SARS-CoV-2 collected in California during the first months of the pandemic were sequenced to define the dynamics of mutation emergence as the virus became established in the state. Deep sequencing of 90 nasopharyngeal samples showed that many mutations associated with the establishment of SARS-CoV-2 globally were present at varying frequencies in a majority of the samples, even those collected as the virus was first detected in the US. A subset of mutations that emerged months later in consensus sequences were detected as subconsensus members of intra-host populations. Spike mutations P681H, H655Y, and V1104L were detected prior to emergence in variant genotypes, mutations were detected at multiple positions within the furin cleavage site, and pre-emergent mutations were identified in the nucleocapsid and the envelope genes. Because many of the samples had a very high depth of coverage, a bioinformatics pipeline, Mappgene, was established that uses both iVar and LoFreq variant calling to enable identification of very low-frequency variants. This enabled detection of a spike protein deletion present in many samples at low frequency and associated with a variant of concern.

Automated summary

Genetic analysis of SARS-CoV-2 positive clinical samples collected in California during the early months of the pandemic revealed insights into the emergence and spread of viral mutations. Many mutations associated with global variants were present at varying frequencies even in samples collected during the initial detection of the virus in the US. Subconsensus mutations emerged later in the consensus sequences. Spike protein mutations and mutations in the furin cleavage site, nucleocapsid, and envelope genes were detected prior to their emergence in variant genotypes. A bioinformatics pipeline enabled detection of low-frequency variants, including a spike protein deletion associated with a variant of concern.