Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance

Sara E. Zufan; Katherine A. Lau; Angela Donald; Tuyet Hoang; Charles S. P. Foster; Chisha Sikazwe; Torsten Theis; William D. Rawlinson; Susan A. Ballard; Timothy P. Stinear; Benjamin P. Howden; Amy V. Jennison; Torsten Seemann

doi:10.1099/mgen.0.001146

Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance

Sara E. Zufan¹, Katherine A. Lau², Angela Donald^1,3, Tuyet Hoang^1,3, Charles S. P. Foster^4,5, Chisha Sikazwe^6,7, Torsten Theis², William D. Rawlinson^2,4,5,8,9, Susan A. Ballard³, Timothy P. Stinear¹, Benjamin P. Howden^1,3, Amy V. Jennison¹⁰ and Torsten Seemann^1,3
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Affiliations: ¹ Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia ² RCPAQAP Biosecurity, St. Leonards, NSW, Australia ³ Microbiological Diagnostic Unit Public Health Laboratory, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia ⁴ Serology and Virology Division (SAViD) SEALS Microbiology, NSW Health Pathology, Sydney, NSW, Australia ⁵ School of Biomedical Sciences, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, Australia ⁶ Department of Microbiology, PathWest Laboratory Medicine Western Australia, Nedlands, WA, Australia ⁷ School of Biomedical Sciences, The University of Western Australia, Nedlands, WA, Australia ⁸ School of Women’s and Children’s Health, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, Australia ⁹ School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, NSW, Australia ¹⁰ Public Health Microbiology, Forensic and Scientific Services, Queensland Department of Health, Brisbane, Australia
*Correspondence: Torsten Seemann, [email protected]
Published: 29 November 2023 https://doi.org/10.1099/mgen.0.001146

Abstract

The COVID-19 pandemic has necessitated the rapid development and implementation of whole-genome sequencing (WGS) and bioinformatic methods for managing the pandemic. However, variability in methods and capabilities between laboratories has posed challenges in ensuring data accuracy. A national working group comprising 18 laboratory scientists and bioinformaticians from Australia and New Zealand was formed to improve data concordance across public health laboratories (PHLs). One effort, presented in this study, sought to understand the impact of the methodology on consensus genome concordance and interpretation. SARS-CoV-2 WGS proficiency testing programme (PTP) data were retrospectively obtained from the 2021 Royal College of Pathologists of Australasia Quality Assurance Programmes (RCPAQAP), which included 11 participating Australian laboratories. The submitted consensus genomes and reads from eight contrived specimens were investigated, focusing on discordant sequence data and findings were presented to the working group to inform best practices. Despite using a variety of laboratory and bioinformatic methods for SARS-CoV-2 WGS, participants largely produced concordant genomes. Two participants returned five discordant sites in a high-Cτ replicate, which could be resolved with reasonable bioinformatic quality thresholds. We noted ten discrepancies in genome assessment that arose from nucleotide heterogeneity at three different sites in three cell-culture-derived control specimens. While these sites were ultimately accurate after considering the participants’ bioinformatic parameters, it presented an interesting challenge for developing standards to account for intrahost single nucleotide variation (iSNV). Observed differences had little to no impact on key surveillance metrics, lineage assignment and phylogenetic clustering, while genome coverage <90 % affected both. We recommend PHLs bioinformatically generate two consensus genomes with and without ambiguity thresholds for quality control and downstream analysis, respectively, and adhere to a minimum 90 % genome coverage threshold for inclusion in surveillance interpretations. We also suggest additional PTP assessment criteria, including primer efficiency, detection of iSNVs and minimum genome coverage of 90 %. This study underscores the importance of multidisciplinary national working groups in informing guidelines in real time for bioinformatic quality acceptance criteria. It demonstrates the potential for enhancing public health responses through improved data concordance and quality control in SARS-CoV-2 genomic analysis during pandemic surveillance.

Received: 18/05/2023
Accepted: 14/11/2023
Published Online: 29/11/2023

Keyword(s): bioinformatics , public health genomics , quality assurance and SARS-CoV-2

Funding

This study was supported by the:

National Health and Medical Research Council (Award MRF9200006)
- Principle Award Recipient: WilliamD. Rawlinson

This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.

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/content/journal/mgen/10.1099/mgen.0.001146

Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance

M Gen 9, 001146 (2023); https://doi.org/10.1099/mgen.0.001146

/content/journal/mgen/10.1099/mgen.0.001146

Microbial Genomics

Volume 9, Issue 11

Short Communication

Open Access

Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance

Abstract

Funding

Supplementary material 1

Supplementary material 2

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