Sequencing of SARS CoV2 in local transmission cases through oxford nanopore MinION platform from Karachi Pakistan

  1. Samina Naz Mukry
  2. Shariq Ahmed
  3. Ali Raza
  4. Aneeta Shahni
  5. Gul Sufaida
  6. Arshi Naz
  7. Tahir Sultan Shamsi

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Abstract

The first case of severe acute respiratory syndrome 2 (SARS CoV2) was imported to Pakistan in February 2020 since then 10,258 deaths have been witnessed. The virus has been mutating and local transmission cases from different countries vary due to host dependent viral adaptation. Many distinct clusters of variant SARS CoV2 have been defined globally. In this study, the epidemiology of SARS CoV2 was studied and locally transmitted SARS CoV2 isolates from Karachi were sequenced to compared and identify any possible variants.The real time PCR was performed on nasopharyngeal specimen to confirm SARSCoV2 with Orf 1ab and E gene as targets. The viral sequencing was performed through oxford nanopore technology MinION platform. Isolates from first and second wave of COVID-19 outbreak in Karachi were compared. The overall positivity rate for PCR was 26.24% with highest number of positive cases in June. Approximately, 37.45% PCR positive subjects aged between 19-40 years. All the isolates belonged to GH clade and shared missense mutation D614G in spike protein linked to increased transmission rate worldwide. Another spike protein mutation A222V coexisted with D614G in the virus from second wave of COVID-19. Based on the present findings it is suggested that the locally transmitted virus from Karachi vary from those reported from other parts of Pakistan. Slight variability was also observed between viruses from first and second wave. Variability in any potential vaccine target may result in failed trials therefore information on any local viral variants is always useful for effective vaccine design and/or selection.

Author’s summary

Despite precautionary measures the COVID-19 pandemic is causing deaths all over the world. The continuous mutations in viral genome is making it difficult to design vaccines. Variability in genome is host dependent and data sharing has revealed that variant for different geographical locations may harbor different mutations. Keeping this in mind the current study was focused on the epidemiology of SARS CoV2 in symptomatic and asymptomatic COVID –19 suspected cases with impact of age and gender. The locally transmitted SARS CoV2 isolates from Karachi were sequenced to compared and identify any possible variants. The sequenced viral genome varied from the already submitted sequences from Pakistan thereby confirming that slightly different viruses were causing infections during different time periods in Karachi. All belonged to GH clade with D614G, P323L and Q57H mutations. The virus from second wave had A222V mutation making it more different. This information can be useful in selecting or designing a vaccine.

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  1. ScreenIT

    SciScore for 10.1101/2021.01.07.425705: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: & Bone Marrow Transplantation after approval from NIBD Bioethics committee.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    For sequencing SQK-LSK104 ligation kit was used and samples were loaded as per standard protocol on a MinION MK1B instrument.
    MinION
    suggested: (MinION, RRID:SCR_017985)
    The acquired data was aligned in real time with SARS CoV2 reference sequence (NC_045512.2) using Min Know Files in SAM/BAM format were accessed by SAM tools v. 1.9-11.
    SAM
    suggested: (SAM, RRID:SCR_010951)
    Further variant annotation was done by ANNOVAR.
    ANNOVAR
    suggested: (ANNOVAR, RRID:SCR_012821)
    The fast alignment was performed using MAFFT.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    The generated tree was visualized using FigTree 1.4.3(http://tree.bio.ed.ac.uk/software/figtree).
    FigTree
    suggested: (FigTree, RRID:SCR_008515)
    For statistical analyses and graphs SPSS version 22 was used.
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    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: 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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • No funding statement was detected.
    • 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 10.1101/2021.01.07.425705v1 on bioRxiv