Whole-Genome Sequencing of Vero E6 (VERO C1008) and Comparative Analysis of Four Vero Cell Sublines
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Abstract
The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of Vero sublines have been developed and can be classified into four major cell lineages. In this study, we determined the whole-genome sequence of Vero E6 (VERO C1008), which is one of the most widely used cell lines for the proliferation and isolation of severe acute respiratory syndrome coronaviruses (SARS-CoVs), and performed comparative analysis among Vero JCRB0111, Vero CCL-81, Vero 76, and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that these four sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we identified four genes ( IL1RAP , TRIM25 , RB1CC1 , and ATG2A ) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoVs, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 harbored only the allele with isoleucine, resulting from the loss of one of the X chromosomes.
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SciScore for 10.1101/2021.10.26.466002: (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 The short-read sequences of Vero JCRB0111, Vero CCL-81, and Vero 76 were downloaded from a public database (DDBJ: PRJDB2865). Vero JCRB0111suggested: NoneVerosuggested: NoneVero 76suggested: NonePaired-end sequences of Vero E6 were determined using an Illumina HiSeq 2500. Vero E6suggested: NoneThe RNA-seq experiment data of Vero E6 TMPRSS2+ cells were downloaded from the public database (SRR13091741–SRR13091746) (Zhang et al. 2021). Vero E6 TMPRSS2+suggested: JCRB …SciScore for 10.1101/2021.10.26.466002: (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 The short-read sequences of Vero JCRB0111, Vero CCL-81, and Vero 76 were downloaded from a public database (DDBJ: PRJDB2865). Vero JCRB0111suggested: NoneVerosuggested: NoneVero 76suggested: NonePaired-end sequences of Vero E6 were determined using an Illumina HiSeq 2500. Vero E6suggested: NoneThe RNA-seq experiment data of Vero E6 TMPRSS2+ cells were downloaded from the public database (SRR13091741–SRR13091746) (Zhang et al. 2021). Vero E6 TMPRSS2+suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Software and Algorithms Sentences Resources The single nucleotide variants (SNVs) and short indels (<50 bp) were called using VarScan 2 software (Koboldt et al. 2012). VarScansuggested: (VARSCAN, RRID:SCR_006849)The GTF format file downloaded from the Ensembl database was used for gene annotation (Chlorocebus_sabaeus.ChlSab1.1.86.gtf) and snpEFF software was used for annotating the effect of each genetic variation (Cingolani et al. 2012). Ensemblsuggested: (Ensembl, RRID:SCR_002344)snpEFFsuggested: (SnpEff, RRID:SCR_005191)The assignment for the gene function categories was performed using DAVID (Jiao et al. 2012). DAVIDsuggested: (DAVID, RRID:SCR_001881)For evaluating the impact of missense variants, we used PROVEAN, SIFT, and PANTHER-PSEP (Choi et al. 2012; Choi and Chan 2015; Tang and Thomas 2016). PROVEANsuggested: (PROVEAN, RRID:SCR_002182)SIFTsuggested: (SIFT, RRID:SCR_012813)The RNA-seq reads were mapped to the reference genome using HISAT2 using the default parameters (Kim et al. 2019). HISAT2suggested: (HISAT2, RRID:SCR_015530)Copy number variations and loss of heterozygosity: We used Control-FREEC software to identify copy number variations (CNVs) and loss of heterozygosity (LOH) (Boeva et al. 2012). Control-FREECsuggested: (Control-FREEC, RRID:SCR_010822)The f2 values were used as a genetic distance between two sublines and a neighbor-joining tree was constructed using MEGA X (Knyaz et al. 2018). MEGAsuggested: (Mega BLAST, RRID:SCR_011920)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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