Figure - available from: Frontiers in Microbiology
This content is subject to copyright.
IGV plot showing seven different amplicons mapped to the SARS-CoV-2 reference genome for four samples with low CT values.
Source publication
Introduction
Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 pri...
Similar publications
Metatranscriptomics is uncovering more and more diverse families of viruses with RNA genomes comprising the viral kingdom Orthornavirae in the realm Riboviria. Thorough protein annotation and comparison are essential to get insights into the functions of viral proteins and virus evolution. In addition to sequence- and hmm profile‑based methods, pro...
Background
Transcriptome data from a plant sample frequently include numerous reads originating from RNA virus genomes that were concurrently isolated during RNA preparation. These high-throughput sequencing reads from the virus can be assembled to form a new sequence for the plant RNA genome.
Methods and results
Here, we identify putative novel m...
The complexity and speed of evolution in viruses with RNA genomes makes predictive identification of variants with epidemic or pandemic potential challenging. In recent years, machine learning has become an increasingly capable technology for addressing this challenge, as advances in methods and computational power have dramatically improved the pe...
Understanding the mechanisms by which single-stranded RNA viruses regulate capsid assembly around their RNA genomes has become increasingly important for the development of both antiviral treatments and drug delivery systems. In this study, we investigate the effects of RNA-induced allostery in a single-stranded RNA virus—Levivirus bacteriophage MS...
Recommendations released by the CDC in 2023 address the need to demonstrate that the RNA genome of positive-strand RNA viruses is inactivated in addition to viral particles. This recommendation is in response to the similarities between host mRNA and the viral genome that allow the viral RNA to be used as a template by host replication mechanisms t...
Citations
... Long amplicon-based multiplex assays only require a small number of primers in each reaction and minimise the chance of introducing too many PCR products amplified differentially in the same tube [30]. Independent studies on SARS-CoV-2 WGS demonstrated that long amplicons-based sequencing not only reduced the possibility of amplicon dropout and coverage bias, but also improved overall quality of the consensus sequences [31,32]. In addition, as the mutation rate is normally high in RNA virus, the larger number of primers used in the amplicon generation method for WGS the higher chances of primer mismatch or drop-out it will have. ...
... This can contribute to uneven coverage of the WGS and may even cause the loss of a whole region of the genome. Significant loss of SARS-CoV-2 genome coverage was reported due to constant viral evolution and especially short amplicon-assays designed with Primal Scheme [31,33]. Therefore, continuous implementation of new primer sets has been used for SARS-CoV-2 surveillance work to restore WGS capability over the entire COVID-19 pandemic period [33,34]. ...
Background
Whole‐genome sequencing (WGS) provides critical insights into the respiratory syncytial virus (RSV) transmission and any emerging mutations that could impair the efficacy of monoclonal antibodies or vaccines that have been recently licenced for clinical use worldwide. However, the ability to sequence RSV genomes at large scale is limited by expensive and time‐consuming sequencing methods. Oxford Nanopore Technology (ONT) offers significant improvements in next generation sequencing (NGS) both in turnaround time and cost, compared with other platforms for viral WGS.
Methods
We have developed and modified an RSV long amplicon‐based WGS protocol for the ONT platform using a one‐step multiplex RT‐PCR assay and the rapid barcoding kit. One hundred thirty‐five RSV positive Australian clinical specimens (91 RSV‐A and 44 RSV‐B) sampled in 2023 with cycle threshold (Ct) values between 14 to 35 were tested in this study. This ONT workflow was compared with other recent RSV WGS amplification assays based on short amplicons.
Results
A PCR amplicon clean‐up step prior to library preparation significantly improved WGS result for samples with poor amplicon generation, but it is not necessary or beneficial for ones that generated high concentrations of amplicons. Overall, a success rate of 85.9% was achieved for WGS. This method performed as well as the more complex short amplicon methods in terms of genome coverage and sequencing depth.
Conclusions
The workflow described here was highly successful in generating RSV WGS on ONT platform and had improved turnaround times and excellent results with RSV clinical samples with Ct values up to 30.
... Long amplicon-based multiplex assays only require a small number of primers in each reaction and minimise the chance of introducing too many PCR products amplified differentially in the same tube (26). Independent studies on SARS-CoV-2 WGS demonstrated that long amplicons-based sequencing not only reduced the possibility of amplicon dropout and coverage bias, but also improved overall quality of the consensus sequences (27,28). In addition, as the mutation rate is normally high in RNA virus, the larger number of primers used in the amplicon generation method for WGS the higher chances of primer mismatch or dropout it will have. ...
... This can contribute to uneven coverage of the WGS and may even cause the loss of a whole region of the genome. Significant loss of SARS-CoV-2 genome coverage was reported due to constant viral evolution and especially short amplicon-assays designed with Primal Scheme (27,29). Therefore, continuous implementation of new primer sets has been used for SARS-CoV-2 surveillance work to restore WGS capability over the entire COVID-19 pandemic period (29,30). ...
Background
Whole-genome sequencing (WGS) provides critical insights into the Respiratory syncytial virus (RSV) transmission and any emerging mutations that could impair the efficacy of monoclonal antibodies or vaccines that have been recently licenced for clinical use worldwide. However, the ability to sequence RSV genomes at large scale is limited by expensive and time-consuming sequencing methods. Oxford Nanopore Technology (ONT) offers significant improvements in next generation sequencing (NGS) both in turnaround time and cost, compared to other platforms for viral WGS.
Methods
We have developed and modified an RSV long amplicon-based WGS protocol for the ONT platform using a one-step multiplex RT-PCR assay and the rapid library barcoding kit. 135 RSV positive Australian clinical specimens (91 RSV-A and 44 RSV-B) sampled in 2023 with Cycle threshold (Ct) values between 14 to 35 were tested in this study. This ONT workflow was compared with other recent RSV WGS amplification assays based on short amplicons.
Results
A PCR amplicon clean-up step prior to library preparation significantly improved WGS result for samples with poor amplicon generation, but it is not necessary or beneficial for ones that generated high concentrations of amplicons. Overall, a success rate of 85.9% was achieved for WGS. This method performed as well as the more complex short amplicon methods in terms of genome coverage and sequencing depth.
Conclusions
The workflow described here was highly successful in generating RSV WGS on ONT platform and had improved turnaround times and excellent results with RSV clinical samples with Ct values up to 30.
... This model determines the necessary coverage threshold to achieve a 50% detection probability, demonstrating a positive correlation between sequencing depth and reliable variant identification. Despite increased sequencing efforts, amplicon sequencing did not reach the 50% efficiency threshold, suggesting that alternative technologies such as shotgun metagenomic sequencing might provide better detection rates through more uniform coverage (Nicot et al. 2023;Kandel et al. 2024). ...
... Benchmarking between probe-based and NGS detection of SARS-CoV-2 from wastewater nucleic acids, in the assessment of SARS-CoV-2 variant detection methodologies, our findings underscore a crucial limitation of standard amplicon sequencing on wastewater samples: its failure to detect all circulating variants can be attributed to the specificity of primers and the variability in sequencing coverage (Johnson et al. 2022;Nicot et al. 2023;Kandel et al. 2024;Lipponen et al. 2024). This specificity often leads to biases in which certain variants may be preferentially amplified or missed, particularly when primer sites undergo mutations. ...
Studies have shown the presence of SARS-CoV-2 in the stool of both symptomatic and asymptomatic COVID-19 patients, enabling wastewater-based surveillance (WBS) to complement clinical monitoring. The emergence of variants can enhance viral transmissibility, highlighting the need for ongoing surveillance to detect and control infectious diseases. This study aimed to detect SARS-CoV-2 variants in wastewater from a treatment plant in San Pedro de la Paz, Chile, between January and November 2021. Wastewater samples were concentrated using the polyethylene glycol method, and RT-qPCR assays were performed to analyze SARS-CoV-2 and its variants (Alpha, Beta, Gamma, Lambda, and Delta), with results compared to Illumina amplicon sequencing. The concentration method achieved about 11% viral recovery. The detection of viruses and variants in wastewater proved sensitive and consistent with clinical data, providing additional surveillance insights. Notably, Lambda and Delta variants were the most frequently detected during the second and third infection waves, with some variants identified in wastewater before the first confirmed clinical cases. However, Illumina sequencing lacked sufficient genome coverage, suggesting the need for better sequencing methods for this matrix. This study demonstrates that WBS is a rapid, cost-effective tool for detecting SARS-CoV-2 and its mutations, particularly useful during overwhelming clinical situations or when cost is prohibitively high.
Accurate pathogen identification is crucial during outbreaks, especially with the emergence of new variants requiring frequent primer updates. However, resources for maintaining up-to-date verification of primer sequences are often limited, which poses challenges for reliable diagnosis and hinders potential monitoring efforts based on genome sequencing. To address this, we introduce ViralPrimer, a web server facilitating primer design, SARS-CoV-2 and Mpox variant monitoring, and adaptation to future threats. ViralPrimer aims to enhance diagnostic accuracy with its comprehensive primer database, mutation analysis assistance, and user primer upload feature. Its adaptable design allows monitoring of other rapidly mutating pathogens, contributing to broader public health protection efforts.
Availability and implementation
ViralPrimer is freely accessible and open to all users with no login requirement at https://viralprimer.elte.hu/. The application is hosted on a DJANGO v3.2.13 web server, with a PostgreSQL database, and the frontend was implemented using jQuery v3.6.0, vanilla JavaScript vES6, and Bootstrap v5.1.
High-throughput sequencing (HTS) has revolutionised the field of pathogen genomics, enabling the direct recovery of pathogen genomes from clinical and environmental samples. However, pathogen nucleic acids are often overwhelmed by those of the host, requiring deep metagenomic sequencing to recover sufficient sequences for downstream analyses (e.g., identification and genome characterisation). To circumvent this, hybrid-capture target enrichment (HC) is able to enrich pathogen nucleic acids across multiple scales of divergences and taxa, depending on the panel used. In this review, we outline the applications of HC in human pathogens—bacteria, fungi, parasites and viruses—including identification, genomic epidemiology, antimicrobial resistance genotyping, and evolution. Importantly, we explored the applicability of HC to clinical metagenomics, which ultimately requires more work before it is a reliable and accurate tool for clinical diagnosis. Relatedly, the utility of HC was exemplified by COVID-19, which was used as a case study to illustrate the maturity of HC for recovering pathogen sequences. As we unravel the origins of COVID-19, zoonoses remain more relevant than ever. Therefore, the role of HC in biosurveillance studies is also highlighted in this review, which is critical in preparing us for the next pandemic. We also found that while HC is a popular tool to study viruses, it remains underutilised in parasites and fungi and, to a lesser extent, bacteria. Finally, weevaluated the future of HC with respect to bait design in the eukaryotic groups and the prospect of combining HC with long-read HTS.
