Novel RT-ddPCR Assays for determining the transcriptional profile of SARS-CoV-2

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   ScreenIT

   Jan 13, 2021

   SciScore for 10.1101/2021.01.12.425991: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   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
   Two primer/probe sets that aligned to all SARS-CoV-2 isolates but had 1 or more mismatch with SARS-CoV and greater than 5 mismatches with MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 were selected for each region (Table 1).	HCoV-NL63 suggested: RRID:CVCL_RW88)
   2.4 Validations using SARS-CoV-2 virion RNA: Vero CCL-81 kidney epithelial cells, derived from Cercopithecus aethiops, were infected with SARS-CoV-2 (Isolate: USA-WA1/2020) …

   SciScore for 10.1101/2021.01.12.425991: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   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
   Two primer/probe sets that aligned to all SARS-CoV-2 isolates but had 1 or more mismatch with SARS-CoV and greater than 5 mismatches with MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 were selected for each region (Table 1).	HCoV-NL63 suggested: RRID:CVCL_RW88)
   2.4 Validations using SARS-CoV-2 virion RNA: Vero CCL-81 kidney epithelial cells, derived from Cercopithecus aethiops, were infected with SARS-CoV-2 (Isolate: USA-WA1/2020) at an MOI of 0.003 (250 000 cells/well).	Vero CCL-81 suggested: None
   The virion standard (1000 copies per 5μL RT) was added to RT reactions with or without cellular RNA from A549 cells (lung epithelial cell line) or donor PBMC (both added at 100ng/μl per RT, or 500ng per ddPCR well).	A549 suggested: None
   Software and Algorithms
   Sentences	Resources
   Droplets were read and analyzed using the QuantaSoft software in the absolute quantification mode.	QuantaSoft suggested: None
   2.6 Assay validations in clinical diagnostic samples from SARS-CoV-2 infected individuals: To investigate the viral transcription profile in clinical samples and determine whether our RT-ddPCR assays correlate with a clinical test, we obtained unused nucleic acid (ranging from 8.25-16.8μL) that remained after extraction by the Abbott m2000 instrument from nasopharyngeal swabs from 3 individuals who tested positive with the Abbott Real Time SARS-CoV-2 assay.	Abbott suggested: (Abbott, RRID:SCR_010477)
   The log-linear relationship between viral load measured by RT-PCR (Abbott Real Time SARS-CoV-2 assay) and RT-ddPCR was determined using GraphPad Prism (version 8.4.1).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Limitations of this study should be acknowledged. In order to test our assays in parallel with published assays (total of 11 assays) in background RNA experiments (Fig. 7), we increased RT reaction volumes from 50-70 μL to 125 μL to accommodate the additional assays. In the absence of background RNA, the efficiency appeared to be higher in the 5070μL RT reactions (Fig. 4-5, >100% efficiency for all assays) than the 125μL reactions (Fig. 7; median efficiency=88% [range: 60-133%]). If the discrepancy is not due to a difference in the actual input of the standard, it is possible that larger reaction volumes lead to less efficiency in reverse transcription. However, for application to patient samples, our core panel of 7 assays (Table 1) is sufficient to provide a detailed view of the transcription profile of SARS-CoV-2, so preparation of RT reactions >70μL will likely be unnecessary. For our study of the viral transcription profile and correlation with the Ct value as determined by the Abbott SARS-CoV-2 Real Time Assay, a limited amount of nucleic acid was available from only a small number of de-identified individuals. Despite this small sample size, we demonstrated both the sensitivity of all assays in our panel and their strong correlation with Ct values in diagnostic specimens. These data allude to potential differences in the transcription dynamics of SAR-CoV-2 during the course of infection and merit further investigation.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 31. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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.

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   Read the original source
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   Version published to 10.1101/2021.01.12.425991v1 on bioRxiv

   Jan 12, 2021