Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform

  1. Elizabeth M. Batty
  2. Theerarat Kochakarn
  3. Arporn Wangwiwatsin
  4. Khajohn Joonlasak
  5. Angkana T. Huang
  6. Bhakbhoom Panthan
  7. Poramate Jiaranai
  8. Krittikorn Kümpornsin
  9. Namfon Kotanan
  10. Wudtichai Manasatienkij
  11. Treewat Watthanachockchai
  12. Kingkan Rakmanee
  13. Anthony R. Jones
  14. Stefan Fernandez
  15. Insee Sensorn
  16. Somnuek Sungkanuparph
  17. Ekawat Pasomsub
  18. Chonticha Klungthong
  19. Thanat Chookajorn
  20. Wasun Chantratita

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Abstract

Genomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.

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    SciScore for 10.1101/2020.06.16.154286: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: All procedures have been approved by Ramathibodi Institutional Review Board (EC approval number: MURA2020/676).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    The pool was sequenced on one MiSeq run using MiSeq v2 chemistry with 250bp paired-end sequencing.
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    Sequencing data QC: To identify human contamination, reads were mapped to both the human GrCH38.p11 and the SARS-CoV-2 genome MN908947.3 using Bowtie2 [15].
    Bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    Coverage per amplicon was determined using bedtools v2.29.2 [21].
    bedtools
    suggested: (BEDTools, RRID:SCR_006646)

    Results from OddPub: Thank you for sharing your data.

    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.

    About SciScore

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  2. Version published to 10.1101/2020.06.16.154286v1 on bioRxiv