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Implementation of cost-efficient high-throughput methods for detection of RNA viruses such as SARS-CoV-2 is a potent strategy to curb ongoing and future pandemics. Here we describe Multiplexed SARS-CoV-2 Quantification platform (McQ), an inexpensive scalable framework for SARS-CoV-2 quantification in saliva samples. McQ is based on the parallel sequencing of barcoded amplicons generated from SARS-CoV-2 genomic RNA. McQ uses indexed, target- specific reverse transcription (RT) to generate barcoded cDNA for amplifying viral- and human-specific regions. The barcoding system enables early sample pooling to reduce hands-on time and makes the approach scalable to thousands of samples per sequencing run. Robust and accurate quantification of viral load is achieved by measuring the abundance of Unique Molecular Identifiers (UMIs) introduced during reverse transcription. The use of homemade reverse transcriptase and polymerase enzymes and non-proprietary buffers reduces RNA to library reagent costs to ~92 cents/sample and circumvents potential supply chain shortages. We demonstrate the ability of McQ to robustly quantify various levels of viral RNA in 838 clinical samples and accurately diagnose positive and negative control samples in a testing workflow entailing self-sampling and automated RNA extraction from saliva. The implementation of McQ is modular, scalable and could be extended to other pathogenic targets in future.

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