SARS-CoV-2 Variant Detection From Wastewater Samples Highlights Importance of Complete Genomic Coverage

Recent findings of known and novel SARS-CoV-2 variants underscore the importance of next-generation-sequencing (NGS)-based epidemiology and surveillance research, alongside the need for full genomic coverage of the SARS-CoV-2 virus for NGS studies.

Wastewater samples are increasingly providing community insights into the rate of COVID infection several days in advance of clinical surveillance, but such samples pose unique challenges for accurate variant identification and monitoring due to sample complexity and low viral titers of SARS-CoV-2.

Wastewater monitoring is possible because SARS-CoV-2 is shed through bodily excretions in similar amounts by symptomatic and asymptomatic individuals. It has been reported that some asymptomatic individuals may even shed SARS-CoV-2 at a higher rate than symptomatic individuals. But because the virus appears at low viral titers in collected wastewater, and the material may be degraded or contaminated, it can be difficult to achieve full genomic coverage from such samples to discern if a certain strain has taken hold in an area or to identify new strains.

Accurately calling variants

Jordan RoseFigura, senior staff scientist at Swift Biosciences, noted that increased genomic coverage of SARS-CoV-2 is an important facet of accurate strain identification to ensure that as many mutations as possible are properly identified, even if the mutation is a genomic loss of information. The locations of mutations within the alpha, beta, and gamma lineages demonstrate the need for full genomic coverage for accurate variant identification (Figure 1).

**Figure 1.** Complete genomic coverage permits accurate identification of SARS-CoV-2 lineages, including variants of concern. The SNAP overlapping primer design ensures that all genomic locations are covered evenly, demonstrated by coverage of mutation locations for the alpha (B.1.17), beta (B.1.351), and gamma (B.1.1.28) lineages shown here.

According to Swift, the Swift Normalase Amplicon SARS-CoV-2 Panel (SNAP) kit provides 99.7% coverage of the SARS-CoV-2 genome. RoseFigura said that the SNAP kit is suitable for low viral titer samples and can be used for wastewater surveillance as well.

To enable variant calling in such samples, the SNAP SARS-CoV-2 panel was developed to be compatible with low inputs, RoseFigura said. According to data from Swift, anywhere from 10 to 1 million viral genome copies are sufficient to generate NGS libraries using the SNAP SARS-CoV-2 panel (Figures 2 and 3).

**Figure 2.** Data from an internal Swift study in which SARS-CoV-2 synthetic template material (Twist Bioscience Cat. No. 102024) was mixed with 10ng UHR RNA (Agilent 740000) and converted into first-strand cDNA using the Superscript IV First-Strand Synthesis System (Thermo Fisher 18091050). cDNA was converted into an NGS library with the Swift SNAP SARS-CoV-2 Kit and sequenced on the Illumina MiniSeq System at 2 x 150 bp. The resulting data was downsampled to 280k reads per sample. The low input protocol enables increased data quality at low viral titers/high Cts.

**Figure 3.** The percent bases at greater than 10X coverage at different read depths. Different copy numbers of the irradiated BEI SARS-CoV-2 control (NR-52287) were mixed with 10ng UHR RNA (Agilent 740000) and converted into first-strand cDNA using the Superscript IV First-Strand Synthesis System (Thermo Fisher 18091050). cDNA was converted into an NGS library with the Swift SNAP SARS-CoV-2 Kit and sequenced on the Illumina MiniSeq System at 2 x 150 bp. Inputs with less than 1,000 copies were subjected to the low input protocol, while the standard protocol was used for high viral copy inputs. The libraries were sequenced on an Illumina MiniSeq at 2 x 150 bp. The resulting data was then downsampled and the percent bases at greater than 10X coverage assessed. Even with 10 copies, the coverage is robust at 50K 2x150 reads.

In addition, Swift’s one-tube, overlapping amplicon assay provides built in redundancy for novel variant discovery. The creation of “super amplicons” maintains genomic coverage, even in the presence of mutations that result in primer dropout (Figure 4). This feature is only found in single-tube workflows; super amplicons do not form in two tube workflows. A novel variant that causes a loss in primer function could result in lost coverage and missed mutations.

**Figure 4.** An illustration showing how a “super amplicon” can rescue coverage in the event of a novel mutation causing a loss of function in a primer, including supporting IGV screenshots from this example. The alpha variant (B.1.1.7) has a deletion that occurs at the 3’ end of a primer binding site in the Swift SARS-CoV-2 panel. The deletion is readily observable in both the adjacent and super amplicons, and coverage in the area is maintained by super amplicons. Data generated by Swift.

According to Swift, the 2 hour SNAP library prep workflow multiplexes as many as 1,536 samples per run on an Illumina sequencer and the proprietary overlapping amplicon design yields 99.7% genomic coverage.

RoseFigura is quoted as saying “This throughput and level of coverage will enable genomic surveillance programs to rapidly expand the number of positive COVID samples sequenced for variant determination while also ensuring that no variants are missed.”

Download the SNAP protocol here.