Genome Sequencing of Sewage Detects Regionally Prevalent SARS-CoV-2 Variants

Abstract

Viral genome sequencing has guided our understanding of the spread and extent of genetic diversity of SARS-CoV-2 during the COVID-19 pandemic. SARS-CoV-2 viral genomes are usually sequenced from nasopharyngeal swabs of individual patients to track viral spread. Recently, RT-qPCR of municipal wastewater has been used to quantify the abundance of SARS-CoV-2 in several regions globally. However, metatranscriptomic sequencing of wastewater can be used to profile the viral genetic diversity across infected communities. Here, we sequenced RNA directly from sewage collected by municipal utility districts in the San Francisco Bay Area to generate complete and nearly complete SARS-CoV-2 genomes. The major consensus SARS-CoV-2 genotypes detected in the sewage were identical to clinical genomes from the region. Using a pipeline for single nucleotide variant calling in a metagenomic context, we characterized minor SARS-CoV-2 alleles in the wastewater and detected viral genotypes which were also found within clinical genomes throughout California. Observed wastewater variants were more similar to local California patient-derived genotypes than they were to those from other regions within the United States or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside California, indicating that wastewater sequencing can provide evidence for recent introductions of viral lineages before they are detected by local clinical sequencing. These results demonstrate that epidemiological surveillance through wastewater sequencing can aid in tracking exact viral strains in an epidemic context.

Keywords: coronavirus; environmental microbiology; genomics; metagenomics.

FIG 1

Characterized viruses detected in enriched and unenriched wastewater metatranscriptomes. The relative abundances of viruses with eukaryotic hosts in the RefSeq database as a percentage of total sequencing reads derived from the sample in Amicon ultrafiltration (viral fractionation) (a) and total RNA column and milk of silica samples (b). All samples were enriched with the Illumina respiratory virus panel. (c) Relative abundances of RefSeq viruses in unenriched metatranscriptomics (left) and the same samples after oligonucleotide enrichment with the Illumina respiratory virus panel. (d) The relationship between the quantity of viral genome copies in 40 μl of purified RNA and SARS-CoV-2 genome completeness (measured in breadth of coverage) for each sample. Samples are colored by extraction methodology, and the size of the point corresponds to the mean SARS-CoV-2 depth of coverage.

FIG 2

SARS-CoV-2 SNVs in wastewater samples. (a) Allele frequencies of SARS-CoV-2 in wastewater metatranscriptomes for each sample. Each point is a SNV by location on the SARS-CoV-2 genome (x axis), and the height of the bar (y axis) is the frequency of the alternative allele (relative to the reference genome EPI_ISL_402124) at that position. Wastewater SNVs are colored based on whether they have previously been observed in clinical samples from California, the United States, or neither. (b) Wastewater SARS-CoV-2 frequencies grouped by whether they have been observed in clinical samples from different regions. Most highly abundant SNVs have been observed previously in California or elsewhere in the United States. (c) SARS-CoV-2 SNVs grouped by the number of wastewater samples observed in (out of seven high-quality samples). Most SNVs that were observed in two or more samples have been observed clinically in California. (d) Multiple hypothesis adjusted (Bonferroni correction) P value distribution of hypergeometric tests for overlap between all wastewater SNVs observed and the variants clinically observed and reported in each location (a county level designation in the United States). Alameda County was the most significant comparison.

FIG 3

Time series of SARS-CoV-2 genotypes in California wastewater compared to patients. (a) Frequencies of two SNVs found in the same viral lineage across California clinical samples (black lines) and within each wastewater sample (orange points). (b) Frequencies of three SNVs found in the same viral lineage across California clinical samples (black lines) and within each wastewater sample (green points). (c) Time series of detection for recurrent wastewater genotypes in clinical samples versus wastewater samples. Each row on the y axis is a SNV, and the presence of a point along the x axis indicates when that SNV was detected in either a clinical sample or a wastewater sample.