Disentangling primer interactions improves SARS-CoV-2 genome sequencing by the ARTIC Network’s multiplex PCR
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (ScreenIT)
Abstract
Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, when sample’s viral load was low, several amplicons, especially amplicons 18 and 76, exhibited low coverage or complete dropout. We have determined that these dropouts were due to a dimer formation between the forward primer for amplicon 18, 18_LEFT, and the reverse primer for amplicon 76, 76_RIGHT. Replacement of 76_RIGHT with an alternatively designed primer was sufficient to produce a drastic improvement in coverage of both amplicons. Based on this result, we replaced 12 primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network’s original (V1) and modified (V3) primer set.
Article activity feed
-
SciScore for 10.1101/2020.03.10.985150: (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. Table 2: Resources
Software and Algorithms Sentences Resources Design of alternative primers: Re-design of the primer nCoV-2019_76_RIGHT was done using PRIMER3 software (Untergasser et al. 2012). PRIMER3suggested: (Primer3, RRID:SCR_003139)Other primers were basically re-designed just by shifting their position several nucleotides toward the 5′ ends, but extension or trimming on either end were applied when the medium dissociation temperature (Tm) predicted by the NEB website tool (https://tmcalculator.neb.com/) were considered too low or … SciScore for 10.1101/2020.03.10.985150: (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. Table 2: Resources
Software and Algorithms Sentences Resources Design of alternative primers: Re-design of the primer nCoV-2019_76_RIGHT was done using PRIMER3 software (Untergasser et al. 2012). PRIMER3suggested: (Primer3, RRID:SCR_003139)Other primers were basically re-designed just by shifting their position several nucleotides toward the 5′ ends, but extension or trimming on either end were applied when the medium dissociation temperature (Tm) predicted by the NEB website tool (https://tmcalculator.neb.com/) were considered too low or high. https://tmcalculator.neb.com/suggested: (NEB Tm calculator, RRID:SCR_017969)The number of insert fragments overlapping each defined small representative region were counted by the co er ge function in the bedtools. bedtoolssuggested: (BEDTools, RRID:SCR_006646)Results from OddPub: Thank you for sharing your code.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.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.
-
