Sequencing SARS-CoV-2 in a malaria research laboratory in Mali, West Africa: the road to sequencing the first SARS-CoV-2 genome in Mali

  1. Antoine Dara
  2. Bouréma Kouriba
  3. Amadou Daou
  4. Abdoul Karim Sangare
  5. Djibril Kassogue
  6. Charles Dara
  7. Abdoulaye Djimde

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Abstract

Next generation sequencing (NGS) has become a necessary tool for genomic epidemiology. Even though the utility of genomics in human health has been proved, the genomic surveillance has never been so important until the COVID 19 pandemic. This has been evidenced with the detection of new variants of SARS-CoV-2 in the United Kingdom, South Africa and Brazil recently using genomic surveillance. Until recently, Malian scientists did not have access to any local NGS platform and samples had to be shipped abroad for sequencing. Here, we report on how we adapted a laboratory setup for Plasmodium research to generate the first complete SARS-CoV-2 genome locally. Total RNA underwent a library preparation using an Illumina TruSeq stranded RNA kit. A metagenomics sequencing was performed on an Illumina MiSeq platform following by bioinformatic analyses on a local server in Mali. We recovered a full genome of SARS-CoV-2 of 29 kb with an average depth coverage of 200x. We have demonstrated our capability of generating a high quality genome with limited resources and highlight the need to develop genomics capacity locally to solve health problems. We discuss challenges related to access to reagents during a pandemic period and propose some home-made solutions.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.05.442742: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    The image was subsequently analyzed using a gel analysis tool ImageJ software [9].
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Quality control was performed using FastQC and Multi-FastQC tools.
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    Subsequently, reads were mapped onto the SARS-CoV-2 reference genome (accession number: MN908947.3) using the Burrows Wheeler aligner (BWA) version 0.7.17 [10] with default parameters.
    BWA
    suggested: (BWA, RRID:SCR_010910)
    Post-mapping file generated as bam file was processed with samtools [11] and the vcfR package to determine the percent of aligned reads, the depth of coverage and mapping quality.
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Library estimation was performed using the CollectInsertSizeMetrics tool version 2.8.1 implemented in Picard.
    CollectInsertSizeMetrics
    suggested: None
    Picard
    suggested: (Picard, RRID:SCR_006525)
    Vcftools was used to generate a variant call file (vcf).
    Vcftools
    suggested: (VCFtools, RRID:SCR_001235)
    SnpEff implemented on Galaxy server as well as Nextclade were used to annotate variants.
    SnpEff
    suggested: (SnpEff, RRID:SCR_005191)
    Galaxy
    suggested: (Galaxy, RRID:SCR_006281)
    We performed de novo assembly of the reads using MEGAHIT software [14].
    MEGAHIT
    suggested: (MEGAHIT, RRID:SCR_018551)
    Shorter contigs (<2000bp) were filtered out and the remaining contigs were queried with BLASTN on the NCBI database to recover the SARS-CoV-2 genome.
    BLASTN
    suggested: (BLASTN, RRID:SCR_001598)
    NCBI
    suggested: (NCBI, RRID:SCR_006472)

    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:
    Our study has some limitations. One of these was the lack of quality control of total RNA before library preparation. For instance, we could not check the quality of the total RNA and did not know if the RNA was degraded or not because we did not have all the reagents required to do so. Decision to sequence the sample was solely based on the quantification of total RNA. Nonetheless, during this pandemic period, access to reagents remains a real challenge, quality control of the total RNA may be bypassed. Another issue was the library size estimation accuracy. Even though, an agarose gel analysis represents an alternative solution to overcome fragment size determination with expensive equipment it may overestimate the fragment size. In summary, we provide the first genome of SARS-CoV-2 sequenced locally in Mali. Our result set a precedent for genomic surveillance of strains circulating in the country. We also offer the sequencing platform that can be used by scientists from neighboring countries. The platform could be used to provide insights in tracking the SAR-CoV-2 transmission dynamics, presence and evolution of variants and data generated could inform health authorities with local data for a better control of the disease.

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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

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  2. Version published to 10.1101/2021.05.05.442742v2 on bioRxiv
  3. Version published to 10.1101/2021.05.05.442742v1 on bioRxiv