C19-SPAR-Seq – a multiplexed, NGS platform for high-throughput detection of SARS-CoV-2

Population scale sweeps of viral pathogens, such as SARS-CoV-2, that incorporate large numbers of asymptomatic or mild symptom patients present unique challenges for public health agencies trying to manage both travel and local spread. Physical distancing is the current major strategy to suppress spread of the disease, but with enormous socio-economic costs. However, modelling and studies in isolated jurisdictions suggest that active population surveillance through systematic molecular diagnostics, combined with contact tracing and focused quarantining can significantly suppress disease spread and has significantly impacted disease transmission rates, the number of infected people, and prevented saturation of the healthcare system. However, reliable systems allowing for parallel testing of 10-100,000s of patients in larger urban environments have not yet been employed.

Here researchers from Mount Sinai Hospital and the University of Toronto describe COVID-19 screening using Systematic Parallel Analysis of RNA coupled to Sequencing (C19-SPAR-Seq), a scalable, multiplexed, readily automated next generation sequencing (NGS) platform that is capable of analyzing tens of thousands of COVID-19 patient samples in a single instrument run. To address the strict requirements in clinical diagnostics for control of assay parameters and output, the researchers employed a control-based Precision-Recall and predictive Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of over 600 patients performed with a specificity of 100% and sensitivity of 91% on samples with low viral loads and a sensitivity of > 95% on high viral loads associated with disease onset and peak transmissibility. This study thus establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

Application of C19-SPAR-Seq to detect SARS-CoV-2

rna-seq

a, Schematic representation of the SARS-CoV-2 with the 5 regions targeted for multiplex C19-SPAR-Seq indicated: RdRP (purple), S receptor binding domain (Srbd) (red), S polybasic cleavage site (Spbs) (light red), E (yellow), and N (orange). b, Schematic of the C19-SPAR-Seq strategy for detecting SARS-CoV-2. cDNA is synthesized using reverse transcriptase (RT) from RNA extracted from clinical samples, subjected to multiplex PCR, then barcoded, pooled and analyzed by next generation sequencing (NGS). c, Analysis of archival NASOP swab eluents by C19-SPAR-Seq. A Proof-of-Concept (PoC) cohort (n = 19) was analyzed by C19-SPAR-Seq, and read numbers for each of the indicated amplicons are presented in a heatmap. Control samples (HEK293T, synthetic SARS CoV-2 RNA) are represented in the left panel, while the right panel shows unsupervised 2D hierarchical clustering of results from negative (blue) and positive (red) patients.

Availability –  the code for demultiplexing and mapping: https://github.com/UBrau/SPARpipe, viral mutation assessment and non-specific amplicon assessment: https://github.com/seda-barutcu/FASTQstitch and https://github.com/seda-barutcu/MultiplexedPCR-DeepSequence-Analysis

Aynaud MM, Hernandez JJ, Barutcu S, et al. (2020) A Multiplexed, Next Generation Sequencing Platform for High-Throughput Detection of SARS-CoV-2. medRXiv [online preprint]. [abstract]

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