ANALYSIS OF SARS-COV-2 GENOME SEQUENCES FROM THE PHILIPPINES: GENETIC SURVEILLANCE AND TRANSMISSION DYNAMICS

  1. Francis A. Tablizo
  2. Carlo M. Lapid
  3. Benedict A. Maralit
  4. Jan Michael C. Yap
  5. Raul V. Destura
  6. Marissa M. Alejandria
  7. Joy Ann Petronio-Santos
  8. El King D. Morado
  9. Joshua Gregor A. Dizon
  10. Jo-Hannah S. Llames
  11. Shiela Mae M. Araiza
  12. Kris P. Punayan
  13. John Mark S. Velasco
  14. Julius Aaron Mejia
  15. Maribell Dollete
  16. Sonia Salamat
  17. Christina Tan
  18. Kristianne Arielle D. Gabriel
  19. Shebna Rose D. Fabilloren
  20. Bernard Demot
  21. Shana F. Genavia
  22. Jarvin E. Nipales
  23. Alessandra C. Sanchez
  24. Haifa L. Gaza
  25. Geraldine M. Arevalo
  26. Coleen M. Pangilinan
  27. Shaira A. Acosta
  28. Melanie V. Salinas
  29. Brian E. Schwem
  30. Angelo D. Dela Tonga
  31. Ma. Jowina H. Galarion
  32. Niña Theresa P. Dungca
  33. Stessi G. Geganzo
  34. Neil Andrew D. Bascos
  35. Eva Maria Cutiongco-de la Paz
  36. Cynthia P. Saloma

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Abstract

The spread of the corona virus around the world has spurred travel restrictions and community lockdowns to manage the transmission of infection. In the Philippines, with a large population of overseas Filipino contract workers (OFWs), as well as foreign workers in the local online gaming industry and visitors from nearby countries, the first reported cases were from a Chinese couple visiting the country in mid-January 2020. Three months on, by mid-March, the COVID-19 cases in the Philippines had reached its first 100, before it exploded to the present 178,022 cases (as of August 20, 2020). Here, we report a genomic survey of six (6) whole genomes of the SARS-CoV-2 virus collected from COVID-19 patients seen at the Philippine General Hospital, the major referral hospital for COVID-19 cases in Metro Manila at about the time the Philippines had over a hundred cases. Analysis of commonly observed variants did not reveal a clear pattern of the virus evolving towards a more infectious and severe strain. When combined with other available viral sequences from the Philippines and from GISAID, phylogenomic analysis reveal that the sequenced Philippine isolates can be classified into three primary groups based on collection dates and possible infection sources: (1) January samples collected in the early phases of the pandemic that are closely associated with isolates from Wuhan, China; (2) March samples that are mainly linked to the M/V Diamond Princess Cruise Ship outbreak; and (3) June samples that clustered with European isolates, one of which already harbor the globally prevalent D614G mutation which initially circulated in Europe. The presence of community-acquired viral transmission amidst compulsory and strict quarantine protocols, particularly for repatriated Filipino workers, highlights the need for a refinement of the quarantine, testing, and tracing strategies currently being implemented to adapt to the current pandemic situation.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.22.20180034: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The study received ethics approval from the University of the Philippines Manila Research Ethics Board with approval number 2020-187-01.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Samples were multiplexed to have at least 10 million sequencing reads per sample and were subsequently demultiplexed using bcl2fastq v2.20.
    bcl2fastq
    suggested: (bcl2fastq , RRID:SCR_015058)
    In both of the described procedures, the tool BWA (Li and Durbin 2009) was used for mapping followed by filtering and conversion from BAM to FASTQ format using Samtools (Li et al. 2009).
    BWA
    suggested: (BWA, RRID:SCR_010910)
    Samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    The chosen assemblies were further refined by scaffolding based on BLAST alignment coordinates against the NCBI reference SARS-CoV-2 sequence (NC_045512.2) using a custom Python script.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Python
    suggested: (IPython, RRID:SCR_001658)
    The overall sequence and structural similarities of the scaffolds with the aforementioned reference sequence were also observed using MAUVE (Darling et al. 2004).
    MAUVE
    suggested: (Mauve, RRID:SCR_012852)
    For surveillance purposes, nucleotide and amino acid sequences for five critical SARS-CoV-2 protein products (RNA-dependent RNA polymerase, spike glycoprotein, membrane glycoprotein, envelope protein, and the nucleocapsid phosphoprotein), were extracted from the annotated scaffolds and aligned using MAFFT (Katoh et al. 2002).
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Manual adjustments to the alignments were made as needed using BioEdit (Hall 1999) to correct for errors in annotation transfer.
    BioEdit
    suggested: (BioEdit, RRID:SCR_007361)
    Maximum likelihood tree reconstruction was finally implemented using RAxML (Stamatakis 2014) with the GTRGAMMA model (as determined via jModelTest2) and 1000 bootstraps for node support.
    RAxML
    suggested: (RAxML, RRID:SCR_006086)
    The six scaffold assemblies from the Philippine isolates, together with 1,083 SARS-COV-2 genome sequences also from GISAID (accessed on March 26, 2020, May 26, 2020, and August 06, 2020), were added to the initial alignment of 246 sequences using MAFFT and subsequently trimmed with TrimAl.
    TrimAl
    suggested: (trimAl, RRID:SCR_017334)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 24. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2020.08.22.20180034v1 on medRxiv