Direct RNA Sequencing Reveals SARS-CoV-2 m6A Sites and Possible Differential DRACH Motif Methylation among Variants

Abstract

The causative agent of COVID-19 pandemic, SARS-CoV-2, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 150 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced thus far are, in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because the incorporation of dNTPs hides RNA base modifications. Here, we present an initial exploration of Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3′-untranslated region. We identified fifteen m6A methylated positions, of which, six are in ORF N. Additionally, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants, we show that variants Beta and Eta have a fourth position C > U change in DRACH at 28,884b that could affect methylation. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.

SciScore for 10.1101/2021.08.24.457397: (What is this?)

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

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
SARS-CoV-2 infections: For SARS-CoV-2 infection, the Vero E6 cell line (ATCC® CRL-1586™) was maintained in Minimum Essential Medium (MEM; Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco).	Vero E6 suggested: None
Software and Algorithms
Sentences	Resources
Runs were performed in MinION (Oxford Nanopore) with flowcell FLO-MIN106 for 40 hours and 1.59 million reads were generated.	MinION suggested: (MinION, RRID:SCR_017985)
T…

SciScore for 10.1101/2021.08.24.457397: (What is this?)

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

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
SARS-CoV-2 infections: For SARS-CoV-2 infection, the Vero E6 cell line (ATCC® CRL-1586™) was maintained in Minimum Essential Medium (MEM; Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco).	Vero E6 suggested: None
Software and Algorithms
Sentences	Resources
Runs were performed in MinION (Oxford Nanopore) with flowcell FLO-MIN106 for 40 hours and 1.59 million reads were generated.	MinION suggested: (MinION, RRID:SCR_017985)
The resulting sam files were converted to bam files and all reads were sorted and indexed according to the reference coordinates using samtools (v-1.13) (Li et al., 2009).	samtools suggested: (SAMTOOLS, RRID:SCR_002105)
Five sequences of each variant isolated and sequenced in Brazil were aligned using the Geneious aligner.	Geneious suggested: (Geneious, RRID:SCR_010519)

Results from OddPub: Thank you for sharing your data.

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.
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.

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

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Direct RNA Sequencing Reveals SARS-CoV-2 m6A Sites and Possible Differential DRACH Motif Methylation among Variants

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