Adapting the Illumina COVIDSeq for Whole Genome Sequencing of Other Respiratory Viruses

Open access publication and protocol on the adapatation of DNA sequencing protocols for use with Influenza A/B, Respiratory Syncytial Virus (RSV), and Rhinovirus

Authors: Nqobile Mthembu, Sureshnee Pillay and Colleagues

Journal: LabMed 2025, 2(4), 19; https://doi.org/10.3390/

Acute respiratory infections (ARIs) continue to pose a major global health threat, particularly among vulnerable populations. These infections often present with similar clinical symptoms, complicating accurate diagnosis and facilitating unmonitored transmissions. Genomic surveillance has emerged as an invaluable tool for pathogen identification and monitoring of such infectious pathogens; however, its implementation is frequently limited by high costs.

The widespread use of highthroughput sequencing during the COVID-19 pandemic has created an opportunity to repurpose existing genomic platforms for broader respiratory virus surveillance.

In this study, we evaluated the feasibility of adapting the Illumina COVIDSeq assay – initially designed for SARS-CoV-2 wholegenome sequencing – for use with Influenza A/B, Respiratory Syncytial Virus (RSV), and Rhinovirus. Positive control samples were processed using two approaches for library preparation: four virus-specific multiple workflows and a combined rapid workflow. Both workflows incorporated pathogenspecific primers for amplification and followed the Illumina COVIDSeq protocol for library preparation and sequencing.

Sequencing quality metrics were analysed, including Phred scores, read length distribution, and coverage depth. The study did not identify significant differences in genome coverage and genetic diversity metrics between workflows.

Genome Detective consistently identified the correct species across both methods. The findings of this study demonstrate that the COVIDSeq assay can be effectively adapted for multi-pathogen genomic surveillance and that the combined rapid workflow can offer a cost- and labourefficient alternative with minimal compromise to data quality.

The described workflow is streamlined and adaptable, incorporating an initial amplification step that facilitates rapid protocol modification for a broad range of viral pathogens across all Illumina sequencing platforms. Its versatility has been demonstrated in various studies: for dengue virus on the NovaSeq 6000, the NextSeq 1000/2000, the MiSeq and the iSeq. We have also adapted COVIDSeq for chikungunya virus on the MiSeq; for HIV drug resistance monitorin; for mpox; and hence can be adapted for a range of pathogens provided the use of highly sensitive primers which can then be run on any Illumina platform.

Our findings indicate that adapting this protocol for other respiratory viruses did not reduce the data quality. This is supported by the comparable Phred scores (>30) observed for both SARS-CoV-2 and other respiratory samples that were library prepared using the COVIDSeq assay, reflecting an error rate of 1 in 1000 base calls, or a base call accuracy of 99.9% . Similar Phred score thresholds (>30) have also been accepted in respiratory virus studies focusing on Influenza [55,56]. Furthermore, this assay was compatible with other respiratory viruses, as we obtained read lengths suggested by the Illumina COVIDSeq Kit (Catalogue No. 20065135, San Diego, CA, USA) for all respiratory samples. Other studies have also successfully adapted COVIDSeq for respiratory viruses like RSV, obtaining highquality genomes and enabling researchers to explore the genetic variability of the seasonal viruses. These standardisation attempts of NGS technology and protocols are essential, as they will allow application in clinical laboratories for routine practice at an affordable cost and play a vital role in diagnosing respiratory diseases, developing more appropriate treatment, and keeping surveillance of emerging viruses in the near future.

OPPOSITE PAGE: Figure 1 – Sample preparation and sequencing of multiple respiratory viruses in multiple workflows, indicated by four tubes, and a combined rapid library preparation workflow, indicated by one tube, the different colours indicating different viruses. (A) indicates the multiple library preparation workflow. (B) indicates the rapid combined library preparation workflow.

Access OPEN publication and protocol: https://doi.org/10.3390/ labmed2040019

Mthembu, N.; Pillay, S.; Musopole, H.T.; Naidoo, S.J.; Msomi, N.; Baye, B.C.; Tshiabuila, D.; Memela, N.Z.; Tombo, T.; de Oliveira, T.; et al.

Adapting the Illumina COVIDSeq for Whole Genome Sequencing of Other Respiratory Viruses in Multiple Workflows and a Single Rapid Workflow. LabMed 2025, 2, 19.