We report the nearly complete genome sequence and the genetic variations of a clinical sample of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) collected from a nasopharyngeal swab specimen from a male patient from Harhoura-Rabat, Morocco. The sequence, which was obtained using Ion Torrent technology, is valuable as it carries a recently described deletion (His69-Val70) and substitution (Asn439Lys).
ABSTRACT
We report the nearly complete genome sequence and the genetic variations of a clinical sample of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) collected from a nasopharyngeal swab specimen from a male patient from Harhoura-Rabat, Morocco. The sequence, which was obtained using Ion Torrent technology, is valuable as it carries a recently described deletion (His69-Val70) and substitution (Asn439Lys).
ANNOUNCEMENT
The pandemic of coronavirus disease 2019 (COVID-19) continues to spread worldwide. The use of genomic data in conjunction with epidemiological data can facilitate early decisions for the control of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belonging to the Betacoronavirus genus and Coronaviridae family (1–3). We used the Ion S5 next-generation sequencing (NGS) technology for whole-genome sequencing (WGS) to detect new mutants that are currently spreading and attracting interest in Europe, mainly in the United Kingdom, and in South Africa (4–6).
A clinical sample of SARS-CoV-2 was collected in December 2020 by BioLife Laboratory (Harhoura-Rabat, Morocco) from a nasopharyngeal swab specimen from a male patient from Harhoura-Rabat, Morocco. RNA extraction was performed using the MagaBio plus virus DNA/RNA purification kit II (BioFlux, China). The patient was initially identified as positive for COVID-19 by reverse transcriptase quantitative PCR and exhibited cycle threshold (CT) values of 22.31, 27.24, and 23.53 for the N, RdRp, and E genes, respectively. Then, the cDNA was prepared using the SuperScript VILO cDNA synthesis kit (Invitrogen, Thermo Fisher Scientific, USA). Fifteen microliters of cDNA was used to prepare a SARS-CoV-2 library by using an Ion AmpliSeq kit for Chef DL8 (Thermo Fisher Scientific). The library was adjusted to 30 pM and then loaded onto an Ion Chef instrument (Thermo Fisher Scientific) for emulsion PCR, enrichment, and loading onto an Ion 530 chip. WGS was performed using the Ion AmpliSeq SARS-CoV-2 research panel designed by Thermo Fisher Scientific for complete viral genome sequencing according to the instructions for use on an Ion GeneStudio S5 Prime Series system.
Raw data were analyzed using Torrent Suite software v5.12.0, and the NGS QC Toolkit v 2.3.3 was used to remove low-quality and short reads. Variant Caller v5.10.1.19 was used to detect variants, compared to the reference genome (Wuhan-Hu-1 strain [GenBank accession number MN908947.3]), and the consensus sequence was generated using IRMAreport v1.3.0.2. The annotation was performed using COVID19AnnotateSnpEff v1.3.0.2, a plugin specifically developed for SARS-CoV-2 that can predict the effect of a base substitution (7).
Our findings allowed us to obtain a SARS-CoV-2 genome of 29,826 bp from 1,392,344 reads; 1,373,947 reads were mapped, covering 98.42% of the total genome with a mean depth of 8,863×. The DNA G+C content was 37.99%. Genetic variant analysis revealed a total of 21 mutations, including 7 synonymous and 10 missense variants (Table 1). The spike harbored the disruptive in-frame deletion known as the His69-Val70 deletion. Moreover, an upstream open reading frame 1ab (ORF1ab) mutation at position 241, an upstream ORF8 mutation at position 27800, and a downstream S mutation at position 29734 were reported (Table 1).
TABLE 1.
Types and effects of identified gene variations
| Residue change | Nucleotide position | Nucleotide change | Variation typea | Gene | Effect |
|---|---|---|---|---|---|
| No change assigned | 241 | c.−25C>T | SNP | ORF1ab | Upstream gene variant |
| p.Leu88Leu | 527 | c.262C>T | SNP | ORF1ab | Synonymous variant |
| p.Phe924Phe | 3037 | c.2772C>T | SNP | ORF1ab | Synonymous variant |
| p.Ile2501Thr | 7767 | c.7502T>C | SNP | ORF1ab | Missense variant |
| p.Tyr2594Tyr | 8047 | c.7782C>T | SNP | ORF1ab | Synonymous variant |
| p.Leu3754Phe | 11527 | c.11262G>T | SNP | ORF1ab | Missense variant |
| p.Met4241Ile | 12988 | c.12723G>T | SNP | ORF1ab | Missense variant |
| p.Pro4715Leu | 14408 | c.14144C>T | SNP | ORF1ab | Missense variant |
| p.Val5112Ile | 15598 | c.15334G>A | SNP | ORF1ab | Missense variant |
| p.His5614Tyr | 17104 | c.16840C>T | SNP | ORF1ab | Missense variant |
| p.Ala5922Ser | 18028 | c.17764G>T | SNP | ORF1ab | Missense variant |
| p.Leu6668Leu | 20268 | c.20004A>G | SNP | ORF1ab | Synonymous variant |
| p.Asn6729Asn | 20451 | c.20187C>T | SNP | ORF1ab | Synonymous variant |
| p.His69_Val70del | 21764 | c.204_209 delACATGT | Deletion | S | Disruptive in-frame deletion |
| p.Asn439Lys | 22879 | c.1317C>A | SNP | S | Missense variant |
| p.Asp614Gly | 23403 | c.1841A>G | SNP | S | Missense variant |
| p.Thr1116Thr | 24910 | c.3348T>C | SNP | S | Synonymous variant |
| p.Ile1130Val | 24950 | c.3388A>G | SNP | S | Missense variant |
| p.Arg150Arg | 26972 | c.450T>C | SNP | M | Synonymous variant |
| No change assigned | 27800 | c.−94C>A | SNP | ORF8 | Upstream gene variant |
| No change assigned | 29734 | c.*4350G>C | SNP | S | Downstream gene variant |
SNP, single-nucleotide polymorphism.
The His69-Val70 deletion (spike N-terminal domain) cooccurring with the Asn439Lys mutation (spike receptor binding domain) in the studied case was not reported in Morocco previously. The His69-Val70 deletion is one of the mutations reported for new emergent lineages primarily identified in the United Kingdom, while Asn439Lys was primally reported in Scotland and is now spreading worldwide. Many studies reported that both mutations enhanced binding affinity for the hACE2 receptor, increasing transmissibility, while showing similar clinical outcomes and in vitro replication fitness, compared to the wild-type strain (8–10).
Data availability.
The consensus sequence generated by IRMAreport v1.3.0.2 was deposited in the GenBank and GISAID databases under the accession numbers MW453084 and EPI_ISL_728353, respectively. The raw reads were deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRR13444960.
ACKNOWLEDGMENTS
This work was carried out under national funding from the Moroccan Ministry of Higher Education and Scientific Research (COVID-19 program) to A.I. This work was also supported by a grant from the Moroccan Institute of Cancer Research and the PPR-1 program to A.I.
We declare no competing interests.
REFERENCES
- 1.Alouane T, Laamarti M, Essabbar A, Hakmi M, Bouricha EM, Chemao-Elfihri MW, Kartti S, Boumajdi N, Bendani H, Laamarti R, Ghrifi F, Allam L, Aanniz T, Ouadghiri M, El Hafidi N, El Jaoudi R, Benrahma H, El Attar J, Mentag R, Sbabou L, Nejjari C, Amzazi S, Belyamani L, Ibrahimi A. 2020. Genomic diversity and hotspot mutations in 30,983 SARS-CoV-2 genomes: moving toward a universal vaccine for the “confined virus”? Pathogens 9:829. doi: 10.3390/pathogens9100829. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Laamarti M, Chemao-Elfihri MW, Kartti S, Laamarti R, Allam L, Ouadghiri M, Smyej I, Rahoui J, Benrahma H, Diawara I, Alouane T, Essabbar A, Siah S, Karra M, El Hafidi N, El Jaoudi R, Sbabou L, Nejjari C, Amzazi S, Mentag R, Belyamani L, Ibrahimi A. 2020. Genome sequences of six SARS-CoV-2 strains isolated in Morocco, obtained using Oxford Nanopore MinION technology. Microbiol Resour Announc 9:e00767-20. doi: 10.1128/MRA.00767-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Laamarti M, Alouane T, Kartti S, Chemao-Elfihri MW, Hakmi M, Essabbar A, Laamarti M, Hlali H, Bendani H, Boumajdi N, Benhrif O, Allam L, El Hafidi N, El Jaoudi R, Allali I, Marchoudi N, Fekkak J, Benrahma H, Nejjari C, Amzazi S, Belyamani L, Ibrahimi A. 2020. Large scale genomic analysis of 3067 SARS-CoV-2 genomes reveals a clonal geo-distribution and a rich genetic variations of hotspots mutations. PLoS One 15:e0240345. doi: 10.1371/journal.pone.0240345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Cusi MG, Pinzauti D, Gandolfo C, Anichini G, Pozzi G, Santoro F. 2020. Whole- genome sequence of SARS-CoV-2 isolate Siena-1/2020. Microbiol Resour Announc 9:e00944-20. doi: 10.1128/MRA.00944-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Jenjaroenpun P, Wanchai V, Ono- Moore KD, Laudadio J, James LP, Adams SH, Prior F, Nookaew I, Ussery DW, Wongsurawat T. 2020. Two SARS-CoV-2 genome sequences of isolates from rural U.S. patients harboring the D614G mutation, obtained using Nanopore sequencing. Microbiol Resour Announc 10:e01109-20. doi: 10.1128/MRA.01109-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Velasco JM, Chinnawirotpisan P, Joonlasak K, Manasatienkij W, Huang A, Valderama MT, Diones PC, Leonardia S, Timbol ML, Navarro FC, Villa V II, Tabinas H Jr, Chua D Jr, Fernandez S, Jones A, Klungthong C. 2020. Coding-complete genome sequences of 23 SARS-CoV-2 samples from the Philippines. Microbiol Resour Announc 9:e01031-20. doi: 10.1128/MRA.01031-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Alessandrini F, Caucci S, Onofri V, Melchionda F, Tagliabracci A, Bagnarelli P, Di Sante L, Turchi C, Menzo S. 2020. Evaluation of the Ion AmpliSeq SARS-CoV-2 research panel by massive parallel sequencing. Genes 11:929. doi: 10.3390/genes11080929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Kemp SA, Meng B, Ferriera IATM, Datir RP, Harvey WT, Collier DA, Lytras S, Papa G, The COVID-19 Genomics UK (COG-UK) Consortium , Carabelli AM, Kenyon J, Lever AML, James LC, Robertson DL, Gupta RK. 2020. Recurrent emergence and transmission of a SARS-CoV-2 Spike deletion H69/V70. bioRxiv 2020.12.14.422555. doi: 10.1101/2020.12.14.422555. [DOI]
- 9.da Silva Filipe A, Shepherd JG, Williams T, Hughes J, Aranday-Cortes E, Asamaphan P, Ashraf S, Balcazar C, Brunker K, Campbell A, Carmichael S, Davis C, Dewar R, Gallagher MD, Gunson R, Hill V, Ho A, Jackson B, James E, Jesudason N, Johnson N, McWilliam Leitch EC, Li K, MacLean A, Mair D, McAllister DA, McCrone JT, McDonald SE, McHugh MP, Morris AK, Nichols J, Niebel M, Nomikou K, Orton RJ, O'Toole Á, Palmarini M, Parcell BJ, Parr YA, Rambaut A, Rooke S, Shaaban S, Shah R, Singer JB, Smollett K, Starinskij I, Tong L, Sreenu VB, Wastnedge E, COVID-19 Genomics UK (COG-UK) Consortium , Holden MTG, Robertson DL, Templeton K, Thomson EC. 2021. Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland. Nat Microbiol 6:112–122. doi: 10.1038/s41564-020-00838-z. [DOI] [PubMed] [Google Scholar]
- 10.Thomson EC, Rosen LE, Shepherd JG, Spreafico R, da Silva Filipe A, Wojcechowskyj JA, Davis C, Piccoli L, Pascall DJ, Dillen J, Lytras S, Czudnochowski N, Shah R, Meury M, Jesudason N, De Marco A, Li K, Bassi J, O’Toole A, Pinto D, Colquhoun RM, Culap K, Jackson B, Zatta F, Rambaut A, Jaconi S, Sreenu VB, Nix J, Zhang I, Jarrett RF, Glass WG, Beltramello M, Nomikou K, Pizzuto M, Tong L, Cameroni E, Croll TI, Johnson N, Di Iulio J, Wickenhagen A, Ceschi A, Harbison AM, Mair D, Ferrari P, Smollett K, Sallusto F, Carmichael S, Garzoni C, Nichols J, Galli M, Hughes J, Riva A, Ho A, Schiuma M, Semple MG, Openshaw PJM, Fadda E, Baillie JK, Chodera JD, ISARIC4C Investigators, ISARIC4C Investigators, COVID-19 Genomics UK (COG-UK) Consortium, Rihn SJ, Lycett SJ, Virgin HW, Telenti A, Corti D, Robertson DL, Snell G. 2021. Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity. Cell 184:1–17. doi: 10.1016/j.cell.2021.01.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The consensus sequence generated by IRMAreport v1.3.0.2 was deposited in the GenBank and GISAID databases under the accession numbers MW453084 and EPI_ISL_728353, respectively. The raw reads were deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRR13444960.