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

Abstract

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.

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies

“…Notably, although H655Y and I1081V appear to be linked, these mutations are not co-located on the spike trimer. Although previous studies have shown that H655Y is frequently detected during passage and in variant genotypes [ 11 , 16 , 32 ], the co-occurrence of I1081V has not been documented. Similarly, mutation I569S was selected for during virus only passage but has not been described in the literature.…”

“…Variants were called using Mappgene, ( https://github.com/LLNL/mappgene ) a SARS-CoV-2 variant calling pipeline for high performance computing environments (HPC) [ 16 ]. Mappgene employs BWA-MEM [ 17 ] for read alignment, iVar 1.3.1 for read trimming/filtering/variant calling, and LoFreq v2.1.5 to call variants [ 18 ].…”

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies

“…Notably, although H655Y and I1081V appear to be linked, these mutations are not co-located on the spike trimer. Although previous studies have shown that H655Y is frequently detected during passage and in variant genotypes (11,16,32), the co-occurrence of I1081V has not been documented. Similarly, mutation I569S was selected for during virus only passage but has not been described in the literature.…”

“…2.9 Bioinformatic analysis. Variants were called using Mappgene, (https://github.com/LLNL/mappgene) a SARS-CoV-2 variant calling pipeline for high performance computing environments (HPC) (16). Mappgene employs BWA-MEM (17) for read alignment, iVar 1.3.1 for read trimming/filtering/variant calling, and LoFreq v2.1.5 to call variants (18).…”