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Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow.

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A new, cost-effective Next-Generation Sequencing (NGS) test offers scalable COVID-19 diagnosis. This molecular test provides accurate results and aids in controlling the pandemic by increasing testing capacity.

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Area of Science:

  • Molecular Biology
  • Virology
  • Genomics

Background:

  • The COVID-19 pandemic caused by SARS-CoV-2 has led to a global surge in cases.
  • The gold-standard diagnostic method, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), faces supply shortages and scalability limitations, particularly for RNA extraction.
  • Brazil and other nations struggle with testing capacity due to reliance on imported supplies and equipment constraints.

Purpose of the Study:

  • To develop a cost-effective, scalable molecular diagnostic test for COVID-19 using Next-Generation Sequencing (NGS).
  • To address the bottleneck in RNA extraction and improve testing throughput compared to RT-qPCR.
  • To create an accompanying online platform for managing and scaling testing.

Main Methods:

  • Development of a single-step RNA extraction and NGS-based molecular diagnostic assay for SARS-CoV-2.
  • Utilized Illumina NextSeq 550 and Novaseq platforms for multiplexed sample analysis.
  • Developed the VarsVID online platform for sample registration, data management, and results display.

Main Results:

  • The NGS-based test demonstrated high scalability, multiplexing 1,536 samples per run on the NextSeq 550 and up to 6,144 on the Novaseq.
  • Achieved a general accuracy of 96% compared to RT-qPCR, increasing to 98% for samples with Cycle Threshold (CT) values below 30.
  • NGS results, presented as fragments per million (FPM), correlated with RT-qPCR CT values, providing semi-qualitative detection.

Conclusions:

  • The developed NGS-based test is a cost-effective and highly scalable alternative for COVID-19 molecular diagnosis.
  • The method overcomes limitations of RT-PCR, enhancing testing capacity, especially in resource-limited settings.
  • The integrated VarsVID platform further supports efficient scaling of diagnostic testing to aid pandemic control.