Early sample tagging and pooling enables simultaneous SARS-CoV-2 detection and variant sequencing

Abstract

Most SARS-CoV-2 diagnostic tests have relied on RNA extraction followed by quantitative reverse-transcription PCR assays (RT-qPCR). 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 dramatically 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 prior to reverse transcription. We validated this assay by applying ApharSeq to more than 500 clinical samples from the Clinical Virology Lab 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. Due to early pooling, we were able to estimate a 10-fold to a 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.