Infectious Disease Research and Testing

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According to WHO, infectious diseases kill over 17 million people a year. Seasonal influenza alone causes annual epidemics that are estimated to result in about 3-5 million cases of severe illness and about 290,000 – 650,000 respiratory deaths. Major diseases such as cholera, malaria, tuberculosis, SARS and Coronavirus can make a deadly comeback in many parts of the world. Traditionally, real-time PCR and antibody-based methods are the main tools for detecting and analyzing infectious agents such as fungi, bacteria and viruses, however, such methods can only focus on a limited number of agents and often times suffer from low assay sensitivity and false negatives. In addition, during viral outbreak, real-time RT/PCR allows identification of the pathogen, but it cannot determine the pathway of the dissemination and clusters of cases due to the lack of the sequence information of infectious agents.

Until now, Next-Generation Sequencing (NGS) is used to obtain partial or full sequences of bacterial or viral genomes and applied to infectious disease research, testing and surveillance. The three main methods based on NGS for pathogen whole-genome sequencing are metagenomics, hybrid captured-based sequencing, and PCR amplicon sequencing. Metagenomics, although reference free, has to sequence both the host DNA and total DNA/RNA from the pathogen so that the sequencing depth and cost required can be very high. Hybrid capture-based sequencing can be less effective in dealing with samples containing very low viral or bacterial genetic material. Compared to the other methods, PCR amplicon sequencing could be an ideal approach mainly because it is highly sensitive and specific against low DNA input, cost-effective and easy to implement in routine laboratory use. The workflow is fast and simple to carry out, and the approach allows researchers and assay developers to focus only on the pathogens being investigated.

CleanPlex® NGS Panels – Simple, Affordable Solution for Infectious Disease Research and Surveillance

CleanPlex Custom NGS Panels are powered by Paragon Genomics’ CleanPlex Technology – an ultra-high PCR amplicon-based targeted sequencing technology for next-generation sequencing (NGS). Scientists or clinicians have the option to add molecular barcodes (unique molecular identifiers – UMIs) into amplicon libraries for more accurate detection of pathogen minor variants via CleanPlex UMI Technology

Researchers pick targets or genes that they would like to interrogate, and our experts design and deliver the custom assays in complete kit format (input to sequencing-ready NGS libraries) in just 4 to 6 weeks. 

infectious disease testing of SARS-CoV-2, COVID-19, EBOLA, HIV, HBV, Influenza via NGS amplicon sequencing

Obtain Complete Viral Genomes from Samples with Low Viral Content

Scientists from Broad Institute worked with Paragon Genomics team to design multiple panels to sequence RNA viruses such as lymphocytic choriomeningitis mammarenavirus (LCMV), Ebola virus (EBOV), Powassan virus (POWV), and Lassa virus 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.

Aaron Lin, PhD; Erica Normandin; Kim Lagerborg, Sabeti Lab, Broad Institute of MIT and Harvard

Sensitive, highly multiplexed sequencing of microhaplotypes from the Plasmodium falciparum heterozygome

Prof. Bryan Greenhouse, Dr. Sofonias Tessema and fellow researchers from Chan Zuckerberg Biohub, University of San Francisco, USA in collaboration with researchers from University of Massachusetts, Brown University, Clinton Health Access Initiative, and Centro de Investigação em Saúde de Manhiça (CISM) of Mozambique demonstrate that targeted NGS sequencing with a custom CleanPlex Panel can be a flexible tool for efficient, low cost, and high-throughput characterization of polyclonal of  P. falciparum infections (Malaria), without the need for WGA. READ PAPER

Using multiplex PCR, we were able to simultaneously sequence 100 heterozygome microhaplotypes and key drug resistance targets in a single reaction with consistent, deep coverage, detecting and quantifying minority alleles in DBS samples down to 10 parasites/μL of blood and outperforming whole genome sequences obtained from 50 times more total reads. Data from laboratory controls and in silico analyses indicate that this approach allows for better estimation of genetic diversity within and genetic relatedness between polyclonal samples when compared with panels of SNPs, making it a promising tool for studying the transmission dynamics of P. falciparum.

Quote from the published paper

Free Custom Panel Consultation

High-Performance Targeted Sequencing Solution for Infectious Disease 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 infectious disease research and surveillance, schedule a free consultation below by entering your email address.

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