Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR

  1. Diyanath Ranasinghe
  2. Tibutius Thanesh Pramanayagam Jayadas
  3. Deshni Jayathilaka
  4. Chandima Jeewandara
  5. Osanda Dissanayake
  6. Dinuka Guruge
  7. Dinuka Ariyaratne
  8. Dumni Gunasinghe
  9. Laksiri Gomes
  10. Ayesha Wijesinghe
  11. Ruwan Wijayamuni
  12. Gathsaurie Neelika Malavige

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

As different SARS-CoV-2 variants emerge and with the continuous evolvement of sub lineages of the delta variant, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, due to limited resources as many resource poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a two-step commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies’ (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using Illumina generated <5%. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. This difference results in a base error probability of 1 in 10 by the ONT and 1 in 1000 for Illumina sequencing platform. Sub-consensus single nucleotide variations (SNV) are highly correlated between both platforms (R 2 = 0.79) while indels appear to have a weaker correlation (R 2 = 0.13). Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number or reads, generated less ambiguous bases and was significantly less expensive than Illumina sequencing technology.

Article activity feed

  1. Version published to 10.1371/journal.pone.0265220
  2. ScreenIT

    SciScore for 10.1101/2021.12.05.21267303: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethical approval for the study was obtained by the Ethics Review Committee of the University of Sri Jayewardenepura.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Fastq and Bam statistics were generated using fastp 2 [14], Nanocomp 3 [15], samtools version 1.9 and in-house scripts.
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    VCF data were processed using vcftools version 0.1.15 and bcftools version 1.7[16].
    vcftools
    suggested: (VCFtools, RRID:SCR_001235)
    Multi-way pileup (mpileup) was made from ONT and Illumina alignments before analyzing with VarScan2[17] for detection of low frequency variants.
    VarScan2
    suggested: (VARSCAN, RRID:SCR_006849)
    All the consensus fasta sequences were aligned to the reference sequence MN908947.3 using MAFFT version 7.487 and a maximum likelihood phylogenetic tree was created using IQ-TREE version 1.6.1 [19] occupying the best fit TIM2+F+R2 model with 1000 bootstrap replicates.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    IQ-TREE
    suggested: (IQ-TREE, RRID:SCR_017254)
    Plots were generated in R version 4.0.1 using ggplot2, dplyr and tidyverse.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    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.

    Results from scite Reference Check: We found no unreliable references.

    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.

  3. Version published to 10.1101/2021.12.05.21267303 on medRxiv