Investigating the Extent of Primer Dropout in SARS-CoV-2 Genome Sequences During the Early Circulation of Delta Variants

Borcard, Loïc; Gempeler, Sonja; Miani, Miguel Angel Terrazos; Baumann, Christian; Grädel, Carole; Dijkman, Ronald; Suter‐Riniker, Franziska; Leib, Stephen L.; Bittel, Pascal; Neuenschwander, Stefan; Ramette, Alban

doi:10.3389/fviro.2022.840952

Cited by 20 publications

(22 citation statements)

References 18 publications

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“…Although genome coverage is an important quality indicator for unbiased sequencing approaches, it can be misleading when a small number of amplicons are overamplified or poly-A is carried through the library preparation and sequenced, as observed here with LB10. Instead, monitoring the mean read depth per amplicon would help participants troubleshoot amplicon drop outs, a common issue with this method that requires optimisation (27)(28)(29). Second, the proximity of discordant SNVs to primers can indicate bioinformatic trimming errors.…”

Section: Discussionmentioning

confidence: 99%

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

Zufan

Lau

Donald

et al. 2023

Preprint

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The capacity to undertake whole genome sequencing (WGS) in public health laboratories (PHLs) has grown rapidly in response to COVID-19, and SARS-CoV-2 genomic data has been invaluable for managing the pandemic. The public health response has been further supported by the rapid upgrade and implementation of laboratory and bioinformatic resources. However, there remains a high degree of variability in methods and capabilities between laboratories. In addition to evolving methodology and improved understanding of SARS-CoV-2, public health laboratories have become strained during surges in case numbers, adding to the difficulty of ensuring the highest data accuracy. Here, we formed a national working group comprised of laboratory scientists and bioinformaticians from Australia and New Zealand to improve data concordance across PHLs. Through investigating discordant sequence data from Australia's first external SARS-CoV-2 WGS proficiency testing program (PTP), we show that most discrepancies in genome assessment arose from intrahost variation. While others could be remedied using reasonable, parsimonious bioinformatic quality control. Furthermore, we demonstrate how multidisciplinary national working groups can inform guidelines in real time for bioinformatic quality acceptance criteria. Provision of technical feedback allows laboratory improvement during a pandemic in real time, enhancing public health responses.

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Section: Discussionmentioning

confidence: 99%

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

Zufan

Lau

Donald

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Although genome coverage is an important quality indicator for unbiased sequencing approaches, it can be misleading when a small number of amplicons are overamplified or poly-A is carried through the library preparation and sequenced, as observed here with LB10. Instead, monitoring the mean read depth per amplicon would help participants troubleshoot amplicon drop outs, a common issue with this method that requires optimization [25][26][27]. Second, the proximity of discordant SNVs to primers can indicate bioinformatic trimming errors.…”

Section: Considerations For Assessing Amplicon Sequencing Datamentioning

confidence: 99%

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

Zufan,

Lau,

Donald

et al. 2023

Microbial Genomics

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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.

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“…Mutations that have resulted in primer drop-outs for VOCs include the G142D (Delta and Omicron) in the 2_Right primer, the 241/243del (Beta) that occurs in the 74_Left primer, and the K417N (Beta) or K417T (Gamma) which occurs in the 76_Left primer 10,11 . The Delta/B.1.1.672 variant has also been associated with ARTIC v3 drop-out of primers 72R and 73L 12 .…”

Section: Introductionmentioning

confidence: 96%

“…The Delta/B.1.1.672 variant has also been associated with ARTIC v3 drop-out of primers 72R and 73L 12 .…”

Section: Introductionmentioning

confidence: 96%

SeqPanther: Sequence manipulation and mutation statistics toolset

San

Wyke

Tegally

et al. 2023

Preprint

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Pathogen genomes harbor critical information necessary to support genomic investigations that inform public health interventions such as treatment, control, and eradication. To extract this information, their sequences are analysed to identify structural variations such as single nucleotide polymorphisms (SNPs) and insertions and deletions (indels) that may be associated with phenotypes of interest. Typically, this involves generating a consensus sequence from raw reads, aligning it to a reference and identifying positions where variations occur. Several pipelines exist to map raw reads and assemble whole genomes for downstream analysis. However, there is no easy-to-use, freely available bioinformatics quality control (QC) tool to explore mappings for both positional codons and nucleotide distributions in mapped short reads of microbial genomes. To address this problem, we have developed a fast and accurate tool to summarise read counts associated with codons, nucleotides, and indels in mapped next-generation sequencing (NGS) short reads. The tool, developed in Python, also provides a visualization of the genome sequencing depth and coverage. Furthermore, the tool can be run in single or batch mode, where several genomes need to be analysed. Our tool produces a text-based report that enables quick review or can be imported into any analytical tool for upstream analysis. Additionally, the tool also provides functionality to modify the consensus sequences by adding, masking, or restoring to wild-type mutations specified by the user. Availability: SeqPanther is available at https://github.com/codemeleon/seqPanther, along with the necessary documentation for installation and usage.

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Investigating the Extent of Primer Dropout in SARS-CoV-2 Genome Sequences During the Early Circulation of Delta Variants

Cited by 20 publications

References 18 publications

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

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

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

SeqPanther: Sequence manipulation and mutation statistics toolset

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