Nanopore metagenomic sequencing for detection and characterization of SARS-CoV-2 in clinical samples

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1. Version published to 10.1371/journal.pone.0259712

   Nov 18, 2021
2. 

   ScreenIT

   Aug 25, 2021

   SciScore for 10.1101/2021.08.13.21261922: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Ethics Approval: This study obtained research ethics board approval from the University of British Columbia (H20-02152). Field Sample Permit: Specimens collected as part of routine testing at VGH and the BCCDC were de-identified and only contained a sample ID number, collection date, Ct, and VOC screening result.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Software and Algorithms
   Sentences	Resources
   Amplified cDNA was purified using a 1:1 ratio of PCR Clean DX beads (Aline Biosciences) and eluted in 50 uL nuclease-free water	Aline Biosciences suggested: None
   Samples were sequenced on FLO-MIN106 flowcells …

   SciScore for 10.1101/2021.08.13.21261922: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Ethics Approval: This study obtained research ethics board approval from the University of British Columbia (H20-02152). Field Sample Permit: Specimens collected as part of routine testing at VGH and the BCCDC were de-identified and only contained a sample ID number, collection date, Ct, and VOC screening result.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Software and Algorithms
   Sentences	Resources
   Amplified cDNA was purified using a 1:1 ratio of PCR Clean DX beads (Aline Biosciences) and eluted in 50 uL nuclease-free water	Aline Biosciences suggested: None
   Samples were sequenced on FLO-MIN106 flowcells on MinION MK1b sequencing devices for 72 hours using MinKNOW (Version 4.2.8, Oxford Nanopore Technologies) with live basecalling disabled.	MinION suggested: (MinION, RRID:SCR_017985)
   Output fastq files were uploaded to BugSeq (version 1.1, database version: RefSeq on Jan 28, 2021) for metagenomic classification (22), and results classification results were visualized in Recentrifuge (23).	RefSeq suggested: (RefSeq, RRID:SCR_003496)
   Analysis from BugSeq outputs and visualizations were performed in RStudio (R version 4.1.0) and Python, with all code available at https://gitlab.com/bugseq/sars-cov-2-nanopore-mngs-performance (28).	BugSeq suggested: None Python suggested: (IPython, RRID:SCR_001658)
   Data Availability: Raw FASTQ data has been uploaded to NCBI Bioproject Accession PRJNA752146.	Bioproject suggested: (NCBI BioProject, RRID:SCR_004801)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Despite limitations in SISPA and Nanopore metagenomic sequencing sensitivity, this approach remains a valuable technique for the detection of pathogens that are novel, unexpected or uncharacterized, and therefore unsuitable for targeted approaches such as RT-qPCR or emerging CRISPR-Cas-based diagnostics, which focus only on known pathogens (35). Unlike these existing diagnostic methods, Nanopore mNGS can theoretically detect any pathogen and co-infections, characterize changes in the site-specific microbiota, and capture the carriage of critical virulence or antibiotic-resistant organisms or genes, all of which can impact patient outcomes. Our approach identified several organisms in the nasopharyngeal microbiota that may cause disease in the lower respiratory tract, consistent with sequencing results from a recent study (30). We also did not detect any viral or atypical bacterial co-infections (Supplementary Table 3), concordant with previous reports of a low prevalence of respiratory co-infection in COVID-19 positive samples (36-38). In support of this finding, our study regions saw a dramatic reduction in incidence of other respiratory viruses (eg., influenza and RSV) and bacterial pathogens over our collection period, thought to be secondary to public health interventions. We additionally assessed the ability of SISPA-based mNGS to classify and assemble complete or partial SARS-CoV-2 genomes from RT-qPCR positive specimens. This method can perform dual diagnostic and mole...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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.

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

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3. 

   Version published to 10.1101/2021.08.13.21261922 on medRxiv

   Aug 23, 2021