Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing , a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.
Article activity feed
-
-
SciScore for 10.1101/2020.10.28.20217778: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Sample material and ethics: The present study includes preliminary investigations and results of a clinical performance study approved by the local Ethic Committee of Vienna (#EK 20-208-0920). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources To test performance across a large number of specimens, we used sample plates from gargle collected in HBSS via our in-house testing pipeline (negative for SARS-CoV2) and spiked in purified RNA obtained from HEK293T cells infected with respective virus … SciScore for 10.1101/2020.10.28.20217778: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Sample material and ethics: The present study includes preliminary investigations and results of a clinical performance study approved by the local Ethic Committee of Vienna (#EK 20-208-0920). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources To test performance across a large number of specimens, we used sample plates from gargle collected in HBSS via our in-house testing pipeline (negative for SARS-CoV2) and spiked in purified RNA obtained from HEK293T cells infected with respective virus strains at a ratio of 1:100 (per gargle volume) or dilutions thereof. HEK293Tsuggested: NoneSoftware and Algorithms Sentences Resources In summary, using crude respiratory specimens as input, a 2-step end-point RT-PCR generates high-specificity and uniform representation of correct amplicons across samples and enables pooling of many samples for analysis by NGS. NGSsuggested: (PM4NGS, RRID:SCR_019164)As proof of principle, we optimized primers for influenza A virus, influenza B virus, and rhinovirus to be combined with our SARS-CoV2 specific SARSeq pipeline. SARSeqsuggested: NoneResults from OddPub: Thank you for sharing your code.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Different SARS-CoV2 detection assays have been optimized over the last few months, each with strengths and limitations. For SARSeq, a potential limitation is the time requirement of the assay. Two PCR reactions must be performed followed by NGS and analysis, so the theoretical minimum time required is around 15 hours. In practice, our tests took at least 24 hours from sample preparation to results. Therefore, SARSeq is not ideally suited for situations where immediate results are required. In such cases, antigen tests42 or RT-LAMP43,44 are superior methods. Rather, SARSeq is ideally suited for regular (e.g., once or twice a week) surveillance of infections in a large scale population, with high sensitivity and specificity (i.e. negligible false positive and false negative rates). SARSeq might also be suitable to test symptomatic persons if a turnaround of 24h for the test itself is acceptable. In addition, SARSeq can be implemented in epidemiology studies to understand the spreading dynamics of infections45 and to investigate interaction between different pathogens across large populations46. However, the main advantage of SARSeq is that the same turnaround time of 15-24 hours can be used to simultaneously test tens of thousands of samples. Therefore, SARSeq complements available diagnostic tests, increasing capacity to enable large-scale monitoring efforts.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
-
