Abstract
Most severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic tests have relied on RNA extraction followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays. Whereas automation improved logistics and different pooling strategies increased testing capacity, highly multiplexed next-generation sequencing (NGS) diagnostics remain a largely untapped resource. NGS tests have the potential to markedly increase throughput while providing crucial SARS-CoV-2 variant information. Current NGS-based detection and genotyping assays for SARS-CoV-2 are costly, mostly due to parallel sample processing through multiple steps. Here, we have established ApharSeq, in which samples are barcoded in the lysis buffer and pooled before reverse transcription. We validated this assay by applying ApharSeq to more than 500 clinical samples from the Clinical Virology Laboratory at Hadassah hospital in a robotic workflow. The assay was linear across five orders of magnitude, and the limit of detection was Ct 33 (~1000 copies/ml, 95% sensitivity) with >99.5% specificity. ApharSeq provided targeted high-confidence genotype information due to unique molecular identifiers incorporated into this method. Because of early pooling, we were able to estimate a 10- to 100-fold reduction in labor, automated liquid handling, and reagent requirements in high-throughput settings compared to current testing methods. The protocol can be tailored to assay other host or pathogen RNA targets simultaneously. These results suggest that ApharSeq can be a promising tool for current and future mass diagnostic challenges.
Highlights
Current methods for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing include a panel of reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests, which are typically applied to nasopharyngeal swab samples [1]
We propose an improved RNA sequencing (RNA-seq) protocol that allows for pooling of barcoded samples before RT, which we called amplicon pooling by hybridization and RNA-seq (ApharSeq)
We demonstrate that cross-sample contamination in this workflow is negligible, and we determined sensitivity to be ~800 to 1600 copies/ml, comparable to existing U.S Food and Drug Administration (FDA)– and European Union (EU)–approved tests [20]
Summary
Current methods for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing include a panel of reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests, which are typically applied to nasopharyngeal swab samples [1]. Samples with cycle threshold (Ct) lower than 35 are typically considered positive in these tests [2, 3] These tests are sensitive and specific, access to qualified labor and specialized equipment and reagents have limited testing capacity at different stages of the coronavirus disease 2019 (COVID-19) pandemic [4, 5]. We propose an improved RNA sequencing (RNA-seq) protocol that allows for pooling of barcoded samples before RT, which we called amplicon pooling by hybridization and RNA-seq (ApharSeq). This workflow is relevant to large-scale testing by reducing labor, reagents, and overall costs by orders of magnitude in these settings. We demonstrate that cross-sample contamination in this workflow is negligible, and we determined sensitivity to be ~800 to 1600 copies/ml, comparable to existing U.S Food and Drug Administration (FDA)– and European Union (EU)–approved tests [20]
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