GENOME SEQUENCES

crossm

Genome Sequences of Six SARS-CoV-2 Strains Isolated in

Morocco, Obtained Using Oxford Nanopore MinION
Technology
Meriem Laamarti,a M. W. Chemao-Elfihri,a Souad Kartti,a Rokia Laamarti,b Loubna Allam,a Mouna Ouadghiri,a Imane Smyej,c

Downloaded from http://mra.asm.org/ on August 26, 2020 by guest

Jalila Rahoui,c Houda Benrahma,c Idrissa Diawara,c Tarek Alouane,a Abdelomunim Essabbar,a Samir Siah,i Mohammed Karra,a
Naima El Hafidi,a Rachid El Jaoudi,a Laila Sbabou,d Chakib Nejjari,e Saaid Amzazi,f Rachid Mentag,g Lahcen Belyamani,h
Azeddine Ibrahimia

a Medical Biotechnology Laboratory (MedBiotech), Bioinova Research Center, Rabat Medical and Pharmacy School, Mohammed V University in Rabat, Rabat, Morocco
b Medical Biotechnology Center, Moroccan Foundation for Science, Innovation & Research (MAScIR), Rabat, Morocco
c National Reference Laboratory, Mohammed VI University of Health Sciences (UM6SS), Casablanca, Morocco
d Research Center of Plants and Microbial Biotechnologies, Biodiversity and Environment, Microbiology and Molecular Biology Team, Faculty of Sciences, Mohammed V
University in Rabat, Rabat, Morocco
e International School of Public Health, Mohammed VI University of Health Sciences (UM6SS), Casablanca, Morocco
f Laboratory of Human Pathologies Biology, Faculty of Sciences, Mohammed V University in Rabat, Rabat, Morocco
g Biotechnology Unit, Regional Center of Agricultural Research of Rabat, National Institute of Agricultural Research, Rabat, Morocco
h Emergency Department, Military Hospital Mohammed V, Rabat Medical and Pharmacy School, Mohammed V University in Rabat, Rabat, Morocco
i
Department of Burns, Mohammed V Military Teaching Hospital/Faculty of Medicine and Pharmacy, Mohammed V University in Rabat, Rabat, Morocco

ABSTRACT Here, we report the draft genome sequences of six severe acute respira-
tory syndrome coronavirus 2 (SARS-CoV-2) strains. SARS-CoV-2 is responsible for the
COVID-19 pandemic, which started at the end of 2019 in Wuhan, China. The isolates
were obtained from nasopharyngeal swabs from Moroccan patients with COVID-19.
Mutation analysis revealed the presence of the spike D614G mutation in all six ge-
nomes, which is widely present in several genomes around the world.

S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is classified within the

subgenus Sarbecovirus and genus Betacoronavirus and was first identified in Wuhan,
China (1), as the causative agent for COVID-19 disease. Since then, the number of
COVID-19 cases has risen dramatically (2).
In Morocco, the first SARS-CoV-2 case was confirmed on 2 March 2020. As of 29 June Citation Laamarti M, Chemao-Elfihri MW, Kartti
S, Laamarti R, Allam L, Ouadghiri M, Smyej I,
2020, the number of cases had reached more than 12,248. To understand SARS-CoV-2 Rahoui J, Benrahma H, Diawara I, Alouane T,
genetic diversity and molecular epidemiology in Morocco, we performed complete Essabbar A, Siah S, Karra M, El Hafidi N, El Jaoudi
genome sequencing using the Oxford Nanopore MinION technology. R, Sbabou L, Nejjari C, Amzazi S, Mentag R,
Belyamani L, Ibrahimi A. 2020. Genome
In this study, we announce the genome sequences of six SARS-CoV-2 strains isolated sequences of six SARS-CoV-2 strains isolated in
from patients in Morocco. The samples were obtained by taking nasopharyngeal swabs Morocco, obtained using Oxford Nanopore
MinION technology. Microbiol Resour Announc
from six patients with COVID-19. The viral RNA was extracted directly from the swab using
9:e00767-20. https://doi.org/10.1128/MRA
the QIAamp viral RNA minikit (Qiagen, Germany), and the Transcriptor first-strand cDNA .00767-20.
synthesis kit (Roche) with random hexamers was used to synthetize the viral cDNA. Editor John J. Dennehy, Queens College
The ARTIC v3 primers were used with the Q5 high-fidelity DNA polymerase (New Copyright © 2020 Laamarti et al. This is an
England BioLabs [NEB], USA) for virus DNA enrichment. Amplicons of 400 bp were open-access article distributed under the terms
of the Creative Commons Attribution 4.0
purified using sample purification beads (SPBs) (Illumina, USA) (3) and then quantified International license.
with a Qubit 3.0 fluorometer and used for library preparation. Address correspondence to Azeddine Ibrahimi,
Sequencing was performed on a MinION MK1C instrument with a ligation sequenc- a.ibrahimi@um5s.net.ma.
Received 6 July 2020
ing kit (catalog number SQK-LSK109) according to a standard protocol (Oxford Nano-
Accepted 17 July 2020
pore Technologies [ONT], UK), and the six samples were multiplexed in one run. The R9 Published 6 August 2020
flow cell was used and run for 2 h.

Volume 9 Issue 32 e00767-20 mra.asm.org 1

Laamarti et al.

TABLE 1 Genome features of six strains of SARS-CoV-2

GenBank NCBI SRA No. of Genome GC content Coverage Mapped
Strain accession no. accession no. raw reads size (bp) (%) (ⴛ) read %a
RMPS-01 MT731285 SRX8633309 71,570 29,903 37.96 870.2 99.93
RMPS-02 MT731292 SRX8633310 185,364 29,903 37.96 2,213.9 98.92
RMPS-03 MT731673 SRX8633311 88,452 29,903 37.96 1,022.5 96.51
RMPS-04 MT731327 SRX8633312 113,813 29,903 37.96 1,291 94.9
RMPS-05 MT731468 SRX8633313 70,565 29,903 37.96 834.5 98.2
RMPS-06 MT731764 SRX8633314 128,615 29,903 37.96 1,291 94.9
a Refers to the percentage of reads from the sequenced sample that align directly to a single region on the reference genome.

The sequence reads generated were between 70,565 and 185,364 (Table 1) of raw data
per sample, with average lengths of 454 bp, 455 bp, 455 bp, 452 bp, 455 bp, and 454 bp for

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strains RMPS-01, RMPS-02, RMPS-03, RMPS-04, RMPS-05, and RMPS-06, respectively.
Raw reads were mapped to a SARS-CoV-2 reference genome under GenBank

A3
South_Africa/174

2.2

B
Indonesia/16

Malaysia/44

Sichuan/55
Malaysia 33

Taiwan/61

NC_04551
Oman/48

Greec 225
4
China/11

Qatar/50
Qatar/51

e/226
France n/34

China/10

Taiwan/6

Kore d/237
6
Japan/

38
Japan/

USA/10

7
e/
Mala ysia/46

Japa /71

Belg a/143
Bos ium/25
Korea/
Gua /72
Mala ia/4

a/39
Gua /42

Greec

6
Ma
Slo

Sri_ zil/78 0
n

Fra nce/2 orea/5

Fra th_K a/57
Un

/43

/26
/231

Finla
Ma Beijin /12

ysia

m
lays /192

32
ven ia/45
ite

y
m

Aus nia
Ken

ia

So Lank
la
d_

US nce/2 32
Ge iwan 82

tr
ia
y

US A/10 33
Ar

s
Ta y/1 0
org /63
Tu

Bra
ab

Tu rus /66

Au A/1 5
rke ia/27 9

u
Be irat ait/4 6

U A / a/9 8
ia
_E Ku tan /37

g/8
nis /2

Ca stra 07
US ad a/6
la

U SA/ 108 2
m w
Ka zak Ital dia/ /13

li
Ka
za hs y/2 14

Tu unis ria/ 12 8
es 0
kh ta 17

B gan 100
T e /1 17
5

n
s n

Ni enin da/

si /2 1
ni ia 17
/3

a/ 68
In dia 218

Sw U

9
In ly/

26
itz ga

g
Ita

e nd 0
So
ut Gr rlan a/1 11 8
n/ 19
h_ e e d 7 ni d/ 36
M
or Afri ce/2 185
/ 9 Be olan ce/2 5
oc c a 2 P ran n/3 8
c
Po o /10 4 F pa /18 /65
Po land /689 9 Ja ain nd 1 7
lan /1 3 Sp aila /26 s/6
Ita d/2 9
9
Th stria /49 8 ate
Mo U ly/2 02 mir
ro SA 14 Au an d/23 b_E
No cco/6 /103 Om lan _Ara
rw 89 Fin ited 4
Sp y/2a 7 Un ia/15 ia/19
Ke ain/1 05 Ind man 12
Be nya/1 87 Ro ia/2 51
lgiu
S m/2 44 tv
La atia/2
Lith pain/1 56 Cro A/104
uan
ia 90 US e/87

A2
Italy /208 Chil rgia/2
29

A1a
DRC /221 Geo rgia/22
8
/117
D
Moro RC/11 Geo nia/239
cco 5 Esto /90
Tunis /6889 Chile 8
ia /8
Tunisi /272 Chile 7
a/274 /7
Chile Chile 5
/7
Belgiu /76 Chile
m 145
Denmar /258 Kenya/ /OUA677-19
k/242 Morocco 0
Italy/219 /18
Morocco Uganda
/RMPS-03
Italy/216
Morocco/68
94 Italy/220
Morocco/6903 Kenya/146
Morocco/6896 Cz Republic/245
Latvia/210 Sri_Lanka/58
Latvia/211 Morocco/6888
Bosnia Spain/181
Benin/111 Lithuania/207
DRC/116 Switzerland/186
DRC/114 Turkey/183
6892 Australia/
Morocco/ Morocco
69
/6890
Morocco 119 Morocco
/6895
DRC/ Moroc /6902
13
D C/1
R Moroc
co/15N
a/97 co/688
Canad 95 Germ
a/ any/22 7
Canad a/93 Brazi 7
d l/81
Cana a/96 Braz
il/82
ad
Can da/94 Pola
a
Can el/27 Om nd/201
an/4
Isra l/25 Gam 7
e Ga bia/1
Isra a/70 m 4
trali /4 Ga bia/1 0
A s gland 8
u m
Mo bia/1 42
En /689 ro
Mo cco 1 4
c co /235 r o /6
ro e Mo cco 899
Mo ranc /203 Ire rocc /690
F y 4
rwa /20 Se land o/690 0
No rway 273 Cy neg /222 5
/
No nisia /230 Cy prus al/17
Tu nce /191 DR pru /24 3
F enia t/120 1
r a
C C/ s/24 6
v p
Slo Egy pt/1 71
2 B roat 118 8
y /2 7 Is eijin ia/2
Eg isia /19 1 Is rae g/9 49
u n nia 10 Ta rae l/17
T a A/ /54 Sp iwa l/19
m S ia 4
Ro U rab /18 ai n/
ud _A o/ /16 02

n/ 60
R rae ani /1

y 18
us l/ a/ 96

A
Sa udi rocc eria S-

i_ rke
i_A ra 68 9

Is om ania 09

9
Sa Mo Nig MP

si 21 19

ud Tu
R om /2 47 5
ra bia 91

a/

Sa
/R

R tvia s/2 sh/

Isr bia /52

19
co

La ypru lade sh/7

Cr Isra el/2 3

3
a /5
oa el/ 9

C ng de h/6
oc

Ba ngla des 906

/ 31
or

Ba ngla co/6
ro Nig nce/ 250
M

5
Ba roc 6
o/R eria/ 234
MP 172

Mo ile/8
Co Israe S-05
ia

Ch ile/8 55
t

Ch nia/2 23
bia 24
US /74

Bo nd/2 5
l/

Ire LY/21 40
US /98
a

Isra A/91

ITA nia/2 0

s
F r

Esto ria/17
Taiw el/23

la
A

Nig an/59
mark n/62

9
Moro USA/9 1

Taiw 13
lom

cco/6 9

Italy il/80
/24
cc

A2
USA 04

Braz 85

e
/102

Braz
Brazi 9
Israel 0
a

Brazi
Israel 0
9

Brazil/
l/2

Republi /22

/2
Czech_
c/243
/3

Australi
Poland/2
Mo

/3

Morocco/RM
Morocco/RM
Norway/206
Algeria/2

Morocco/6901
Israel/26
Israel/18
Morocco/RMPS-06
Israel/28
Bosnia/253
Israe

il/
Algeria/1
Algeria

l/83
l/7
Den

84
Republi
a/73

A2A
00
Czech_

PS-01
PS-04

c/244

FIG 1 Phylogenetic tree of six SARS-CoV-2 genomes from Morocco. We are currently sequencing more genomes from Morocco to further investigate the
spread of COVID-19 and to monitor the evolution of SARS-CoV-2 in Morocco.

Volume 9 Issue 32 e00767-20 mra.asm.org 2

Microbiology Resource Announcement

accession number NC_045512.2 using BWA-MEM v. 0.7.17 for single-end reads with
default settings (4), and SAM/BAM files were manipulated by SAMtools v. 1.9-11 (5).
Variant calling was performed using BCFtools v. 1.9 with “mpileup” (5), and variants
were further annotated using SnpEff v. 4.3T (6). The consensus sequences were
generated by mapping the variants to the reference genomes using BCFtools (5) and
then were submitted to the GISAID database and NCBI (accession numbers are listed in
Table 1).
The phylogenetic analysis was realized using 250 genome sequences retrieved from
the GISAID database. The alignment was performed using MAFFT (7) for fast alignment,
and maximum-likelihood trees were inferred with IQ-TREE v. 1.5.5 under the general-
ized time-reversable (GTR) model (8), implemented via the pipeline provided by Augur
(github.com/nextstrain/augur). The generated tree was visualized using FigTree 1.4.3

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(http://tree.bio.ed.ac.uk/software/figtree). Major clades were defined by amino acid
and/or nucleotide substitutions and were matched to the Nextstrain nomenclature (9)
(https://nextstrain.org/ncov).
The size of the consensus sequences was similar to that of the Wuhan-Hu-1
reference genome (GenBank accession number NC_045512.2) and was 29,903 bp with
a mean coverage ranging from 843.5⫻ to 2,213⫻. The strain details are found in
Table 1.
We detected 16 different variants in the 6 analyzed genomes. All the genomes
shared four mutations, namely, two synonymous (F924F and L4715L), one nonsynony-
mous (D614G), and one intergenic (241C⬎T). Only one nonsynonymous mutation was
detected (D614G) in the spike protein, which is known as the most prevalent variant
worldwide (10), and it is also associated with the emergence of clade A2, which
includes all Moroccan strains sequenced in this study (Fig. 1). This mutation was
already associated with the observed transmission increase in the United States
(10–12).
Data availability. The reads of the six SARS-CoV-2 strains were deposited in
DDBJ/ENA/GenBank under the SRA accession numbers SRR12109250, SRR12109251,
SRR12109252, SRR12109253, SRR12109254, and SRR12109255. The consensus se-
quences were also deposited in GenBank under the accession numbers MT731285,
MT731292, MT731673, MT731327, MT731468, and MT731764.

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.

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