Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

  1. John R Tyson
  2. Phillip James
  3. David Stoddart
  4. Natalie Sparks
  5. Arthur Wickenhagen
  6. Grant Hall
  7. Ji Hyun Choi
  8. Hope Lapointe
  9. Kimia Kamelian
  10. Andrew D Smith
  11. Natalie Prystajecky
  12. Ian Goodfellow
  13. Sam J Wilson
  14. Richard Harrigan
  15. Terrance P Snutch
  16. Nicholas J Loman
  17. Joshua Quick

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing ( https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w ) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to £10 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.09.04.283077: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Flowcell costs were based on individual pricing for Flongle and for flowcells packs of 48 (MinION/GridION) and 12 (PromethION).
    PromethION
    suggested: (PromethION, RRID:SCR_017987)

    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).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Here we have addressed several limitations of the ARTIC multiplex PCR method. To eliminate systematic dropouts we introduced the V3 primer scheme which showed the highest genome coverage and lowest CV over a wide range of input cDNA. Modifications to the library preparation protocol have improved the rate of demultiplexing from 42% to 75% while delivering a 35x reduction in the number of incorrectly assigned reads. Further changes to the library preparation protocol reduce the hands-on time by 1-2 hours. Modelling the reagent costs and run configurations allowed us to determine that running 24-48 barcodes on a MinION flowcell is the most cost-effective current run mode with rapid turnaround time. The availability of 96 barcodes allows further cost reduction and makes use of the higher output PromethION instrument cost-effective. The ARTIC SARS-CoV-2 sequencing protocol has been adopted by many groups around the world due to the low cost, high sensitivity compared to other methods. PCR-based target enrichment produces a high rate of on-target products with an inexpensive and simple laboratory workflow. The main disadvantage is that genome coverage is lost for regions that fail to amplify in the multiplex PCR. Itokawa et al. identified a 10bp overlap between two primers in the initial V1 primer scheme which resulted in the loss of two regions due to heterodimer formation. The scheme was successfully modified but further work developing design tools such as PrimalScheme are need...

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.09.04.283077v1 on bioRxiv