Applications
Coronavirus Research and Surveillance

COVID-19 Coronavirus (SARS-CoV-2) Research and Surveillance

Paragon Genomics has developed PCR amplicon-based NGS research panels for both Illumina and MGI Tech Sequencing platforms. The panels are designed to obtain complete viral genomes from samples with low SARS-CoV-2 (the virus responsible for COVID-19) viral content. 

Panel design is based on the SARS-CoV-2 sequence NC_045512.2. A total of 343 primer pairs, distributed in two pools, were selected by a proprietary panel design pipeline to cover the whole genome except for 92 bases at the ends. Primers were optimized to preferentially amplify the SARS-CoV-2 cDNA versus the background human cDNA or DNA. They were also optimized to uniformly amplify the covered genome. Amplicon sizes range from 116bp to 196bp, with a median size of 149 bp. This expertly designed panel allows for the interrogation of the entire viral sequence with as little as 250 thousand reads per samples.

As the virus mutates, the panel contains sufficient targeted regions such that detection will not be affected. With sequencing, these mutations can also be tracked to study the path of infection and examine different strains.

To evaluate performance and sensitivity, the panel has been tested against reference plasmids: pUC backbone containing either the nucleic capsid (N) gene or the spike (S) gene, two surface proteins specific to SARS-CoV-2.Targets were spiked into cDNA converted from Human Total RNA (agilent), and serially diluted down to zero copies per sample and tested to determine LoD. Results show higher sensitivity than traditional qPCR methods (1).
(1).  Victor M Corman et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988269/

Image: Texas Children’s Hospital Department of Pathology- Coronavirus Webinar

The CleanPlex Coronavirus Panel workflow includes 3 simple steps that convert viral RNA to sequencing-ready libraries in 5 hours. CleanPlex® SARS-CoV-2 panel is powered by Paragon Genomics’ CleanPlex Technology, which uses a proprietary multiplex PCR background cleaning chemistry to effectively remove non-specific PCR products, resulting in best-in-class target enrichment performance and efficient use of sequencing reads

Step 1 starts with isolated viral RNA input for reverse transcription. Paragon Genomics’s RT reagents utilize a special mixture of optimized random primers to select for RNA of interest and minimize transcription of other non targeted materials such as rRNA from human cells.

Step 2 consists of the powerful multiplex PCR technology from Paragon Genomic’s CleanPlex product line to simultaneously, uniformly, and efficiently amplify all regions of interest from SARS-CoV-2 genome. The reagents are optimized for ultra-high multiplex PCR for maximum uniformity and minimal nonspecific product formation.

(for Illumina sequencing, an additional 15 minutes purification step  is needed to ensure clean and high quality libraries)

Step 3 is an indexing PCR that allows the addition of index primers for sequencing on either MGI or Illumina sequencing platforms.

We will provide update on our development progress on this page and please stay tuned. To request more information or collaboration, please fill out the form below. 

Free Coronavirus Research Custom NGS Panel Consultation

High-Performance Amplicon Sequencing Solution for Coronavirus (SARS-CoV-2) Research and Surveillance

CleanPlex Custom NGS Panels offer excellent performance and high level of customization to help you build smarter NGS assays. To create a custom panel for your coronavirus research or surveillance projects, schedule a free consultation below by entering your email address.

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Scientists from Broad Institute’s Sabeti Lab worked with Paragon Genomics team to design a panel to sequence RNA viruses such as Ebola using CleanPlex and CleanPlex UMI technologies. The custom NGS assay features a single-tube workflow that is easy to perform and minimizes the risk for handling errors. 

“We’re using Paragon’s CleanPlex and CleanPlex UMI technologies to sequence RNA viruses. These technologies enable us to rapidly and cost-effectively obtain complete viral genomes from clinical samples with low viral content. Further analyses of viral genomes, including the identification of minor variants, elucidates viral diversity and evolution. Paragon has enthusiastically worked with us to tackle this new application; they have diligently designed panels to our specifications, meeting with us frequently to optimize the design.” – Scientists from Broad Institute’s Sabeti Lab