key: cord-0775721-uraysilv
authors: Rfaki, Abderrazak; Touil, Nadia; Hemlali, Mouhssine; Alaoui Amine, Sanaâ; Melloul, Marouane; El Alaoui, Moulay Abdelaziz; Elannaz, Hicham; Lahlou, Amine Idriss; Elouennass, Mostafa; Ennibi, Khalid; El Fahime, Elmostafa
title: Complete Genome Sequence of a SARS-CoV-2 Strain Sampled in Morocco in May 2020, Obtained Using Sanger Sequencing
date: 2021-05-20
journal: Microbiol Resour Announc
DOI: 10.1128/mra.00387-21
sha: a8076bd5803719c4e20c64e7a3e016ff1743d5fb
doc_id: 775721
cord_uid: uraysilv

The complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain was obtained. The strain was isolated from a nasopharyngeal swab specimen from a female patient in Rabat, Morocco, with coronavirus disease 2019 (COVID-19). This strain belongs to clade 20A and has 12 mutations and 8 amino acid substitutions compared to the reference strain Wuhan/Hu-1/2019.

S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a member of the Coronaviridae family and Betacoronavirus genus, is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. As of April 2020, positive cases rose continually, reached a peak in November 2020, and then decreased gradually until now (http://www.covidmaroc.ma/pages/Accueilfr.aspx). Analysis of the SARS-CoV-2 genome is crucial for fully understanding its spread and evolution as well as for vaccine development (1) . Currently, SARS-CoV-2 whole-genome sequences are often generated by next-generation sequencing (NGS) (2), because of the fast speed and parallelism of the standard NGS methods; however, Sanger sequencing can also provide high-quality genome sequences (3) .

Here, we report the complete genome sequence of SARS-CoV-2 from a Moroccan patient, using Sanger sequencing technology, based on multiple amplified nucleic acid fragments, according to CDC (4) protocol.

Strain SARS-CoV-2/human/MAR/Morocco_HMIMV_279CC/2020 was recovered from a nasopharyngeal swab specimen from a 48-year-old female patient in Rabat, Morocco, with COVID-19 on 10 May 2020. Viral RNA was extracted using a MagPurix instrument (Zinexts Life Science, UK) and quality controlled using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Germany). cDNA was generated using a Tetro cDNA synthesis kit (Bioline, UK), and real-time PCR (RT-PCR) was performed using MyFi mix (Bioline) according to the manufacturer's protocol.

The whole-genome sequence of strain SARS-CoV-2/human/MAR/Morocco_HMIMV_ 279CC/2020 was generated using 38 pairs of specific primers covering the whole virus genome using a previously reported method (4) . The PCR amplification yielded 38 amplicons, which were confirmed by gel electrophoresis analysis (1% agarose) and purified using an ExoSAP-IT PCR cleanup kit (Applied Biosystems, USA). The sequence reactions were performed using the BigDye terminator cycle sequencing kit v3.1 (Applied Biosystems), purified using the BigDye XTerminator purification kit (Applied Biosystems), and then loaded onto a SeqStudio genetic analyzer capillary sequencer (Applied Biosystems) according to the manufacturer's instructions.

The obtained nucleotide sequences were evaluated, edited, and assembled, based on mapping to the reference sequence of the SARS-CoV-2 Wuhan-Hu-1 isolate (GenBank accession number MN908947.3) using Unipro UGENE software v37.0. The length of the final consensus sequence is 29,903 nucleotides with 37.96% GC content. NCBI BLASTN v2.11.01 (5) showed that the genome was mostly similar (99.97%) to SARS-CoV-2/human/USA/CA-CZB-1229/2020 (MT499204.1) and to SARS-CoV-2/human/ TWN/CGMH-CGU-15/2020 (MT374111.1), reported in the USA and Taiwan, respectively (Fig. 1) .

Sequence quality control, clade assignment, and mutation determination were done using the Nextclade tool v0.12.0 (https://clades.nextstrain.org/). All tools were run using default parameters unless otherwise specified. The results showed that the SARS-CoV-2/human/MAR/Morocco_HMIMV_279CC/2020 genome belongs to clade 20A, with 12 mutations (A2568T, C3037T, C5884T, C8169T, C9907G, C14408T, C17104T, A20268G, G21795T, A23403G, G25563T, and G29734C) and 8 amino acid substitutions (ORF1a, Q768L; ORF1a, S2635L; ORF1a, Y3214; ORF1b, P314L; ORF1b, H1213Y; ORF3a, Q57H; S, R78M; and S, D614G), without amino acid deletions or PCR primer changes.

Data availability. The complete nucleotide sequence has been deposited in NCBI GenBank under accession number MW582699 and in the GISAID database under identifier EPI_ISL_971451. (7), and the phylogenetic tree was constructed using MEGA X v10.2.0 (8) with the neighbor-joining algorithm with 1,000 bootstrap replicates. The tree structure was rooted in the position of reference strain Wuhan-Hu-1. Bar, number of nucleotide substitutions per site.

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Deep sequencing: becoming a critical tool in clinical virology

Sequencing the complete genome of COVID-19 virus from clinical samples using the Sanger method

A greedy algorithm for aligning DNA sequences

MAFFT multiple sequence alignment software version 7: improvements in performance and usability

The Galaxy platform for accessible, reproducible and collaborative biomedical analyses

MEGA X: molecular evolutionary genetics analysis across computing platforms

This study was supported and financed by the Hassan II Academy of Science and Technology, Morocco. The study was approved by the Biomedical Research Ethics Committee of Mohammed V University, Faculty of Medicine and Pharmacy, Rabat, Morocco (approval number 17/20, delivered on 12 June 2020).