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

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.

SUBMITTER: Chappleboim A

PROVIDER: S-EPMC9928115 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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

Chappleboim Alon A Joseph-Strauss Daphna D Rahat Ayelet A Sharkia Israa I Adam Miriam M Kitsberg Daniel D Fialkoff Gavriel G Lotem Matan M Gershon Omer O Schmidtner Anna-Kristina AK Oiknine-Djian Esther E Klochendler Agnes A Sadeh Ronen R Dor Yuval Y Wolf Dana D Habib Naomi N Friedman Nir N

Science translational medicine 20211103 618

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 v ...[more]

PMID: 34591660

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Project description:BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing. To overcome the challenge of nucleic acid (NA) extraction and testing kit availability, an efficient method is urgently needed.ObjectivesTo establish an efficient, time and resource-saving and cost-effective methods, and to propose an ad hoc pooling approach for mass screening of SARS-CoV-2.MethodsWe evaluated pooling approach on both direct clinical and NA samples. The standard reverse transcriptase polymerase chain reaction (RT-PCR) test of the SARS CoV-2 was employed targeting the nucleocapsid (N) and open reading frame (ORF1ab) genomic region of the virus. The experimental pools were created using SARS CoV-2 positive clinical samples and extracted RNA spiked with up to 9 negative samples. For the direct clinical samples viral NA was extracted from each pool to a final extraction volume of 200μL, and subsequently both samples tested using the SARS CoV-2 RT-PCR assay.ResultsWe found that a single positive sample can be amplified and detected in pools of up to 7 samples depending on the cycle threshold (Ct) value of the original sample, corresponding to high, and low SARS CoV-2 viral copies per reaction. However, to minimize false negativity of the assay with pooling strategies and with unknown false negativity rate of the assay under validation, we recommend pooling of 4/5 in 1 using the standard protocols of the assay, reagents and equipment. The predictive algorithm indicated a pooling ratio of 5 in 1 was expected to retain accuracy of the test irrespective of the Ct value samples spiked, and result in a 137% increase in testing efficiency.ConclusionsThe approaches showed its concept in easily customized and resource-saving manner and would allow expanding of current screening capacities and enable the expansion of detection in the community. We recommend clinical sample pooling of 4 or 5 in 1. However, we don't advise pooling of clinical samples when disease prevalence is greater than 7%; particularly when sample size is large.

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

Publications

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

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