Sequencing SARS-CoV-2 genomes from saliva

Abstract

Genomic sequencing is crucial to understanding the epidemiology and evolution of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Often, genomic studies rely on remnant diagnostic material, typically nasopharyngeal (NP) swabs, as input into whole-genome SARS-CoV-2 next-generation sequencing pipelines. Saliva has proven to be a safe and stable specimen for the detection of SARS-CoV-2 RNA via traditional diagnostic assays; however, saliva is not commonly used for SARS-CoV-2 sequencing. Using the ARTIC Network amplicon-generation approach with sequencing on the Oxford Nanopore MinION, we demonstrate that sequencing SARS-CoV-2 from saliva produces genomes comparable to those from NP swabs, and that RNA extraction is necessary to generate complete genomes from saliva. In this study, we show that saliva is a useful specimen type for genomic studies of SARS-CoV-2.

Keywords: SARS-CoV-2; saliva; genomic epidemiology; next generation sequencing; oxford nanopore MinION; salivadirect.

Figure 1.

Saliva performs comparably to nasopharyngeal (NP) swabs as an original sample for SARS-CoV-2 genome sequencing. (A) The percent of genome with at least 20× coverage is plotted against the Ct value for the N1 target for a cohort of unpaired saliva (blue) and NP swab (yellow) samples. Samples with a Ct value ≤30 (vertical black dashed line) and a genome completeness <80 per cent (horizontal grey line) are displayed in panel B. (B) The percent of the genome at different coverage thresholds (legend, top right) is plotted against Ct value for the N1 target for select samples from A. Grey lines connect points related to the same sample. (C) A subset of samples from the cohorts in panel A are plotted against the number of reads for each sample, showing that nearly all samples (saliva and NP swab) with at least 200,000 reads (vertical black line) have >80 per cent genome completeness. The mean readcount for each cohort is displayed underneath the legend.

Figure 2.

SARS-CoV-2 genomes from matched saliva and NP swabs are similar in completeness and content. (A) A cohort of matched saliva and NP swab samples from the same individual were sequenced and reads were subsampled to match the mate with fewer reads. A grey line connects the mates and an empty circle highlights the mate with lower coverage. (B) A maximum-likelihood tree of matched saliva and NP swab samples from Fig. 2A is rooted against the reference genome (NCBI Accession MN908947.3) to show pairwise identity. Tips of the tree aligning vertically indicate that the genomes from these samples are identical.

Figure 3.

RNA extraction dramatically improves SARS-CoV-2 genome completeness from saliva samples. (A) Saliva samples were split to perform either RNA extraction (blue) or SalivaDirect lysate (brown) preparation (incubation with Proteinase K at 95°C for 5 minutes; see Section 4) and were sequenced. The percent of genome with at least 20× coverage is plotted against the Ct value for the N1 target for matched samples (connected by grey line). (B) The percent of the genome at different coverage thresholds (legend, right) is plotted against Ct value for the N1 target for the same cohort of samples in panel A. Grey lines connect points related to the same sample.