The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) around the world, causing successive waves of coronavirus disease 2019 (COVID-19), has led to intensive research on SARS-CoV-2 , as well as its detection and treatment. One of the most important threads has been the need to detect new and emerging variants of SARS-CoV-2, as they are often associated with greater transmission and disease severity.
Amplicon-based sequencing methods have been widely used in this regard. Yet the evidence for their usefulness is still lacking in completeness and strength.
Study: Clinical Performance Characteristics of the Swift Normalase Amplicon Panel for Sensitive Recovery of SARS-Cov-2 Genomes. Image Credit: Zita / Shutterstock.com
Millions of SARS-CoV-2 genomes are publicly available for analysis, using next-generation sequencing platforms that enable genomic monitoring under near real-world conditions. This includes whole genome sequencing (WGS) of viral isolates from COVID-19 positive patients, which play a major role in the investigation of local epidemics, intervention planning, development of preventive and therapeutic pharmaceutical agents. , as well as the evaluation of the effectiveness of these drugs.
The variants of concern of SARS-CoV-2 (VOCs) are new variants linked to higher transmissibility, reduced vaccine efficacy and adverse outcomes, usually due to specific sets of mutations that lead to immune evasion and improved viral replication. Again, sequencing is essential to identify such mutations and develop monoclonal antibodies. Finally, the emergence of new strains in immunocompromised patients with chronic COVID-19 can be monitored by such technologies.
Multiplexed amplicon sequencing is preferred over shotgun sequencing or capture-based sequencing methods for its increased sensitivity, speed and low cost. Previously, the authors of the present study published on the medRxiv * Preprint server demonstrated that such a panel could recover genomes up to cycle threshold (Ct) on many isolates.
The Swift SNAP primer, the subject of this study, is designed to complement the ancestral Wuhan-Hu-1 genome and amplifies 345 amplicons across the entire ribonucleic acid (RNA) strand. This primer takes three hours or less to build amplicon libraries, of only 10 to 100+ copies of the virus. Further processing can be done manually or enzymatically using proprietary formulations.
Due to the lack of guidelines for evaluating WGS tests for SARS-CoV-2, the present study used the United States Food and Drugs Administration (FDA) guidelines to validate SARS-CoV-2 One-tube Swift Normalase Amplicon Panel (SNAP). Version 2.0 of Swift Biosciences.
This is the first time that such validation has been performed on this commercially available test which has been used in a number of vaccine trials and research studies.
The researchers found they were able to recover high quality genomes from COVID-19 positive samples with a 95% detection limit of 40 copies of SARS-CoV-2 by polymerase chain reaction (PCR) ( PCR), at concentrations of 82 copies / reaction or Continuation. The genomes were recovered over a range of Ct values of approximately 11 to 37, with a median value of 22.
The researchers did not recover genomes from samples negative by PCR or positive for other viruses. They obtained pairwise identity for the 100% consensus sequences in all cases.
Replicated genomes recovered on the same or different cycles were of good quality, as the allele frequencies were highly matched between the Swift and shotgun sequencing libraries. The use of different instruments also made it possible to achieve a high degree of reproducibility. The researchers further found that both VOC and non-VOC strains were correctly identified, using reference data sets from the U.S. Centers for Disease Control and Prevention (CDC).
Mixtures of different SARS-CoV-2 clades over a range of ratios led to detection of the expected strains even at a ratio of 1:99. Once released for clinical use, the test was used almost 270 times in the first 23 weeks. The median turnaround time was 11 days, and its use was primarily for investigating outbreaks and controlling infections.
Allelic frequency measured (y-axis) for all expected mutations (n = 20) in the sample mixes described in Table 2. Blue panels = mutations common to samples 20A and 20B, orange panels = mutations expected in sample 20B, purple panel = mutations expected in sample 20A.
The results of the present study confirm that multiplex PCR amplifier panels can be used to perform high throughput sequencing of SARS-CoV-2. Not only can they be used to retrieve genomes across a broad spectrum of viral loads, they also reduce workflow time.
Validation of this platform using FDA guidelines for other NGS tests in this study shows that it can be used in routine clinical work in any laboratory approved by the Clinical Laboratory Improvement Amendments (CLIA ) and the College of American Pathologies (CAP). The approach discussed here was both very sensitive, with a limit of detection of whole genomes at 40 copies per reaction, and was found to accurately recover allele frequencies among mixed samples.
This is essential to assess the variation of the SARS-CoV-2 lineage within the same host, as can occur after vaccination or treatment. In fact, amplicon sequencing may be more relevant to capture alleles with minor frequencies compared to capture-based sequencing technologies.
The Swift SNAP panel can be used directly with short read technologies as it produces amplicons of less than 255 base pairs (bp) compared to others like ARTIC, which, although inexpensive, produce amplicons of more of 400 bp. This means that additional fragmentation is required before using Illumina based sequencing or Oxford Nanopore Technologies (ONT) based direct sequencing.
Since more primers are used in this panel, as well as overlapping amplicons, the dropouts of amplicons are relatively low. Another advantage is the ability of this panel under optimized parameters recommended by the manufacturer to recover genomes over a range of Ct values, as in real specimens.
The availability of an optional proprietary enzymatic normalization step (Normalase) can produce equimolar pools also covering a range of samples and amplicons, making it easier to manage samples in large batches rather than adding more cycles of d ‘amplification. The latter can lead to low quality analyzes due to excessive dimerization of the primers, as well as the amplification of trace contaminants.
“Our results demonstrate the high sensitivity, specificity and reproducibility of the Swift SNAP Amplicon Panel for SARS-CoV-2, making it ideal for clinical applications.”
Researchers provide a link to the protocol at https://www.protocols.io/view/uw-virology-swift-snapv2-protocol-byw4pxgw. This can be used with robotic systems and is applicable for the validation of other amplicon sequencing methods.
medRxiv publishes preliminary scientific reports that are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.