SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing

  1. AS Speranskaya
  2. VV Kaptelova
  3. AV Valdokhina
  4. VP Bulanenko
  5. AE Samoilov
  6. EV Korneenko
  7. OY Shipulina
  8. VG Akimkin

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Abstract

Here we provide technical data for amplifying the complete genome of SARS-CoV-2 from clinical samples using only seventeen pairs of primers. We demonstrate that the СV2000bp primer panel successfully produces genomes when used with the residual total RNA extracts from positive clinical samples following diagnostic RT-PCRs (with Ct in the range from 13 to 20). The library preparation method reported here includes genome amplification of ~1750-2000 bp fragments followed by ultrasonic fragmentation combined with the introduction of Illumina compatible adapters. Using the SCV2000bp panel, 25 complete SARS-CoV-2 virus genome sequences were sequenced from clinical samples of COVID-19 patients from Moscow obtained in late March - early April.

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    SciScore for 10.1101/2020.08.04.234880: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationSubsequently, the primer combinations were optimized using iterative sequencing of some randomly selected samples on Illumina MiSeq.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    The quality and fragment length distribution of the obtained libraries were evaluated with Agilent Bioanalyzer 2100 (Agilent Technologies, USA).
    Agilent Bioanalyzer
    suggested: None
    In short, reads were trimmed by quality with Trimmomatic [7].
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    PCR primers were removed using Cutadapt [8].
    Cutadapt
    suggested: (cutadapt, RRID:SCR_011841)
    Resulting reads were mapped to reference genome MT121215.1 using bowtie2 [9].
    bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    Reads with low mapping quality were filtered out using SAMtools [10].
    SAMtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Basecalling was performed with GATK [11].
    GATK
    suggested: (GATK, RRID:SCR_001876)
    Gvcf files were filtered with BCFtools [12], and the consensus sequence was obtained with BEDTools [13].
    BCFtools
    suggested: (SAMtools/BCFtools, RRID:SCR_005227)
    BEDTools
    suggested: (BEDTools, RRID:SCR_006646)

    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:
    • 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.

    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 10.1101/2020.08.04.234880v1 on bioRxiv