Combining short and long read sequencing technologies to identify SARS-CoV-2 variants in wastewater

Pillar 5
Viral Genomics and Sequencing

Wastewater surveillance is used to track COVID-19 infection levels in a community, as well as the types of SARS-CoV-2 variants present.  As the SARS-CoV-2 genome continues to evolve, it becomes difficult to identify new and distinct variants in a mixed population sample such as wastewater. Next-generation sequencing technologies, such as Illumina and Nanopore, provides rapid results with high throughput, and have been a highly effective tool for wastewater surveillance. This study investigates the advantages of combining Illumina and Nanopore sequencing for the purpose of improving detection and characterization of SARS-CoV-2 variants in wastewater samples. 

248 wastewater samples were collected from various cities in Quebec and Ontario.  Each sample was processed to extract and amplify SARS-CoV-2 RNA, and the resulting PCR products (amplicons) were sequenced using both Illumina and Nanopore technology.  Illumina sequencing resulted in fragmented sequences of base pairs (reads) that were of higher quality and were sequenced at a higher depth, resulting in a high accuracy output, while Nanopore sequencing resulted in longer reads that can provide more complete information about certain regions of the genome.

Key findings:

• Mutations that were preferentially detected by Illumina sequencing were most common in variant lineages that were of low abundance or unknown in the population for that time period.
• Nanopore sequencing had a higher detection rate of mutations commonly found in the B.1.1.7. (Alpha) variant, which was the most abundant variant at the time. It also resulted in a higher sequencing rate of mutations that are in the same amplicon, which can be a more robust indicator of the presence of variants, such as Alpha, which have key mutations close together.
• Combining the datasets of Illumina and Nanopore sequencing resulted in increased detection of variants of concern (VOCs) and variants of interest (VOIs).

This study highlights the effectiveness of both short-read Illumina sequencing and long-read Nanopore sequencing towards detecting distinct SARS-CoV-2 variants and presents a workflow that combines both sequencing methods to improve detection of variants in wastewater. 

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