COVID-19 Coronavirus (SARS-CoV-2) Sequencing for Detection and Surveillance

Go to the CleanPlex SARS-CoV-2 Panel sequencing performance section.

Paragon Genomics has developed PCR amplicon-based NGS panels for sequencing the whole genome of SARS-CoV-2 (the virus responsible for COVID-19) on both Illumina and MGI Tech Sequencing platforms. The panels are designed to obtain complete viral genomes even from samples with very low SARS-CoV-2 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 200 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 during our initial development phase, the panel was 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/

After we launched our products in late March, we have received sequencing data from several customers who tested our panel on positive patient samples. The results showed that our panel can detect low viral copies and obtain almost full genome information of the virus even at low sequencing depths. See the performance data here.

CoronaVirus SARS-CoV-2 Structure

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

CleanPlex RNA SARS-CoV-2 Amplicon Sequencing library preparation and target enrichment workflow

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.

Paragon Genomics’ CleanPlex SARS-CoV-2 NGS Panel shows exceptional sensitivity on very low virus copy numbers even at a low sequencing depth. Data below are obtained from our hospital collaborators using CleanPlex Panel to sequence patient samples that were tested positive by RT-PCR. With a high-throughput NGS sequencer from Illumina or MGI, you can pool hundreds to thousands of samples onto a single sequencing run.

To ensure that our customers get the most out of their sequencing runs, we’re proud to offer up to 2688 sample multiplexing capability with our combinatorial dual-indexed primers for Illumina sequencing. Additionally, we provide 384 unique-dual indexes, allowing for low variant calling and other in-depth sequencing applications. These exciting additions allow for cost-effective and high-throughput sequencing with our best-in-class CleanPlex targeted sequencing technologies.

CleanPlex SARS-CoV-2 Panel Sensitivity for Low Virus Copy Number at 1.7M Reads

Down sampling was performed in silico for samples with various viral loads. All sequencing metrics maintains high performance even at 50,000 total paired-end (PE) reads at even down to 20 viral copies/rxn. 

CleanPlex SARS-CoV-2 genome coverage vs viral copy number at different sequencing depths
CleanPlex SARS-CoV-2 Panel uniformity at 0.2x mean after down sampling
CleanPlex SARS-CoV-2 Panel mapping rate after down sampling
CleanPlex SARS-CoV-2 Panel on target rate after down sampling
customer testimonial broad institute logo

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

CleanPlex Coronavirus Research Consultation