sherzad ppt.pptx

Whole Genome sequence analysis of SARS-COV-2 isolated
from COVID-19 patients in Duhok, Iraq.
Dr.Sherzad M Taher
Medical Immunology
December 1,2022

Introduction
• The recent appearance of the Severe Acute Respiratory
Syndrome Coronavirus 2 (SARS-CoV-2) has attracted global
attention and prompted an international campaign to develop
a treatment and vaccine.
• The outbreak began in Wuhan, China, in December 2019 and
has since spread to every country and impacting virtually
everyone in the world.
• Since November 2022, there have been 646,998,989
confirmed cases of COVID-19, with 6,638,678 deaths
reported.

Introduction
• The SARS-CoV-2 virus is an enveloped single-stranded
positive-sense RNA (+ ssRNA), encoding around 9860 amino
acids .Typical SARS-CoV-2 has one of the largest genomes
∼ 30 kb, among all RNA virus families.

The shape of the SARS-CoV-2 genome

Introduction cont..
• Viral genome sequences from different countries can assist in
determining the virus's mutational tendencies as it crosses
diverse geographical environments and peoples of various
cultures, with differing genetic profiles and immunological
responses.

Aim of Study
• The main aim of this study was to carry out whole-genome
sequencing (WGS) of the local SARS-CoV2 variant to gain
insights into the mutational variation of the virus compared to
the first reported genome sequence of the SARS-COVID-2
genome isolated from Wuhan/China.
• In addition, this sequencing enabled a precise understanding
of the novel variations, which are key indicators for viral
development and vaccine response.

Clinical sample and processing
 A nasopharyngeal swab collected from patients with respiratory
tract infection in Duhok received at the Covid-19 Center/ UOD for
six months for COVID-19 testing.
 The selected samples were subjected to extraction and
quantitative detection using RNA extraction and real-time PCR
 Ct value of selected samples was (< 20)
 The RNA was then extracted from the viral transport medium
transported to the USA on dry ice for whole genome sequencing.

Genomic sequencing
• The samples were retested for target confirmation and RNA
integrity following arrival at USA .
• Nanomere primers from were used for reverse transcription and cDNA
synthesis.
• Next-generation sequencing was performed using illumina instrument and
equipment(Illumina,,USA) following manufacturers ‘instructions was used
to evaluate sequencing data.
• The obtained whole-genome sequence was submitted to the GISAID
database, and the assigned accession numbers were received.

The assigned accession numbers submitted to the GISAID
database
• EPI_ISL_12604438 , EPI_ISL_12604442 , EPI_ISL_12604444 , EPI_ISL_12604448
,EPI_ISL_12604451,EPI_ISL_12604457 EPI_ISL_12604460 ,EPI_ISL_12604463,
EPI_ISL_12604471,EPI_ISL_12604476 EPI_ISL_12604477, EPI_ISL_12604478
,EPI_ISL_12604481, EPI_ISL_12604482 ,EPI_ISL_12604483,EPI_ISL_12604487
,EPI_ISL_12604488,EPI_ISL_12604489, EPI_ISL_12604490 , EPI_ISL_12604495,
EPI_ISL_12604496,EPI_ISL_12604501 ,EPI_ISL_12604502,EPI_ISL_12604503
,EPI_ISL_12604507, EPI_ISL_12604508 EPI_ISL_12604509, EPI_ISL_12604510
,EPI_ISL_12604514 ,EPI_ISL_12604516 ,EPI_ISL_12604517, EPI_ISL_12604521 ,
EPI_ISL_12604526, EPI_ISL_12604527 ,EPI_ISL_12604528,EPI_ISL_12604532,
EPI_ISL_12604845, EPI_ISL_12604846 ,EPI_ISL_12604847, EPI_ISL_12604848

Genome assembly, alignment, and phylogenetic
analysis
• The software Nextstclade and ClustalW were used to analyze the
uniqueness of the sequenced genomes and to align full sequences
of the genome to the SARS-CoV-2 reference genome (NC 045512.2)
• A phylogenetic analysis was done using Molecular Evolutionary
Genetics Analysis version (MEGA 11.0) to examine similarities and
differences between the Duhok genome sequences and other
SARS-CoV-2 genome sequence downloaded from GenBank and the
GISAID database.

No. Upload Acc. ID Dist. Qual. Accession ID Collect. date Subm. date Lin. Country / State
1 EPI_ISL_12604438 1 0.923 EPI_ISL_10185095
1
2022-02-01 2022-02-22 BA.1.1 United Kingdom / England
2 EPI_ISL_12604442 0 0.988 EPI_ISL_12604518
2
2022-02-10 2022-05-09 BA.1.1 Iraq / Kurdistan / Duhok
3 EPI_ISL_12604444 0 0.984 EPI_ISL_12604847
2
4 EPI_ISL_12604448 1 0.977 EPI_ISL_8295166
3
2021-12-25 2022-01-03 BA.1.1 Singapore
5 EPI_ISL_12604451 0 0.928 EPI_ISL_10249214
1
2022 2022-02-23 BA.1.1 United Kingdom / England
6 EPI_ISL_12604457 1 0.902 EPI_ISL_10557396
4
2022-02-09 2022-03-02 BA.1.1 USA / Colorado
7 EPI_ISL_12604460 2 0.952 EPI_ISL_13711474
5
2022-01-04 2022-07-08 BA.1.1 Kyrgyzstan / Chui
8 EPI_ISL_12604463 1 0.970 EPI_ISL_12604423
2
9 EPI_ISL_12604471 0 0.903 EPI_ISL_9224622
6
2021-12-30 2022-01-28 BA.1 Germany / North Rhine-Westphalia
10 EPI_ISL_12604476 2 0.916 EPI_ISL_12058290
7
2021-12-19 2022-04-14 BA.1.1 USA / California / Placer County
11 EPI_ISL_12604477 0 0.982 EPI_ISL_12604462
2
12 EPI_ISL_12604478 0 0.955 EPI_ISL_9222066
6
2022-01-16 2022-01-28 BA.1.1 Germany / Hamburg
13 EPI_ISL_12604481 0 0.921 EPI_ISL_9021360
8
14 EPI_ISL_12604482 0 0.987 EPI_ISL_12604485
2
15 EPI_ISL_12604483 0 0.977 EPI_ISL_13528076
9
2022-02-02 2022-06-28 BA.1.1 Austria / Styria / Liezen
16 EPI_ISL_12604487 0 0.954 EPI_ISL_9830331
10
2022-01-20 2022-02-12 BA.1 Poland / Swietokrzyskie Voivode-
ship
17 EPI_ISL_12604488 0 0.915 EPI_ISL_8456340
8
2021-12-28 2022-01-07 BA.1.1 United Kingdom / Scotland
18 EPI_ISL_12604489 1 0.966 EPI_ISL_10230026
11
2022-01-22 2022-02-22 BA.1 Poland / Lodzkie / Lodz
19 EPI_ISL_12604490 0 0.974 EPI_ISL_10676026
12
2022-02-11 2022-03-04 BA.1.1 United Kingdom / Wales
20 EPI_ISL_12604495 1 0.919 EPI_ISL_11955489
1
21 EPI_ISL_12604496 1 0.918 EPI_ISL_10042180
4
2022-02-02 2022-02-17 BA.1.1 USA / Illinois
22 EPI_ISL_12604501 0 0.961 EPI_ISL_12604518
2
23 EPI_ISL_12604502 0 0.980 EPI_ISL_12604412
2
24 EPI_ISL_12604503 0 0.973 EPI_ISL_9765953
13
2022-01-06 2022-02-10 BA.2 Canada / British Columbia
25 EPI_ISL_12604507 1 0.971 EPI_ISL_11041477
8
2022-03-07 2022-03-15 None United Kingdom / Scotland
26 EPI_ISL_12604508 0 0.965 EPI_ISL_10011583
14
2021-12-28 2022-02-17 BA.1.1 Kenya / Migori
27 EPI_ISL_12604509 1 0.925 EPI_ISL_8333825
15
2021-12-20 2022-01-04 BA.1.1 Australia / New South Wales / Syd-
ney
28 EPI_ISL_12604510 0 0.967 EPI_ISL_7908939
16
2021-12-10 2021-12-21 BA.1.1 Denmark / Syddanmark
29 EPI_ISL_12604514 2 0.976 EPI_ISL_10416884
8
30 EPI_ISL_12604516 0 0.978 EPI_ISL_12604847
2
31 EPI_ISL_12604517 0 0.978 EPI_ISL_10497679
4
2022-01-11 2022-03-01 BA.1.1 USA / Michigan
32 EPI_ISL_12604521 0 0.968 EPI_ISL_12604516
2
33 EPI_ISL_12604526 1 0.972 EPI_ISL_9283690
8
34 EPI_ISL_12604527 1 0.965 EPI_ISL_9998212
8
2022-02-10 2022-02-17 BA.1.1 United Kingdom / Scotland
35 EPI_ISL_12604528 1 0.975 EPI_ISL_12599035
17
2022-03-31 2022-05-09 BA.1.1 USA / North Dakota
36 EPI_ISL_12604532 2 0.964 EPI_ISL_9233949
6
2021-12-29 2022-01-28 BA.1.1 Germany / Rhineland-Palatinate
37 EPI_ISL_12604845 1 0.924 EPI_ISL_10240172
18
2021-12-22 2022-02-23 BA.1.1 Lithuania / Kauno apskritis
38 EPI_ISL_12604846 0 0.990 EPI_ISL_12604443
2
39 EPI_ISL_12604847 0 0.986 EPI_ISL_12604482
2
2

Total mutations, their locations, and their effects on the changes of the amino acid sequence
within the SARS-COV-2 genome isolated from Duhok /Iraq
Gen Position Nucl.change Code Amino acid Change Type of Mutation
ORF1a (266...13468)
444 GTT > GCT V 60 A Valin>Alanine Non-synonymous SNV *
593 CAT > TAT H 110 Y Histidine>Tyrosine Non-synonymous SNV *
670 AGT > AGG S 135 R Serine>Arginine Non-synonymous SNV *
1415 CTT > TTT L 384 F Leucine>Phenylalanine Non-synonymous SNV *
2790 ACT > ATT T 842 I Threonine>Isoleucine Non-synonymous SNV *
2832 AAG > AGG K 856 R Lysine>Arginine Non-synonymous SNV *
2883 TGT > TAT C 873 Y Cisteine>Tyrosine Non-synonymous SNV *
3896 GTT > TTT V 1211 F Valine>Phenylalanine Non-synonymous SNV *
4184 GGT > AGT G 1307 S Glycine>Serine Non-synonymous SNV *
4893 ACA > ATA T 1543 I Threonin>Isoleucine Non-synonymous SNV *
5007 ACG > ATG T 1581 M Threonin>Methionine Non-synonymous SNV *
510 - 518 ATG > -TG del82/84 del82/84 Non-frame shift deletion
519 ATG > -TG M 85 V Methionine>Valine Non-synonymous SNV
6176 GAT > AAT D 1971 N Aspartic acid>Asparagine Non-synonymous SNV
6513 - 6515 del2083/2083 del2083/2083 Non-synonymous SNV
6516 TTA > -TA L 2084 I Leucine>Isoleucine Non-synonymous SNV
7036 TTA > TTT L 2257 F Leucine>Phenylalanine Non-synonymous SNV
7488 ACT > ATT T 2408 I Threonine>Isoleucine Non-synonymous SNV
8393 GCT > ACT A 2710 T Alanine>Threonin Non-synonymous SNV
9344 CTT > TTT L 3027 F Leucine>Phenylalanine Non-synonymous SNV
9474 GCT > GTT A 3070 V Alanine>Valine Non-synonymous SNV
9866 CTT > TTT L 32201 I Leucine>Isoleucine Non-synonymous SNV
10029 ACC > ATC T 3255 I Threonin>Isoleucine Non-synonymous SNV
10323 AAG > AGG K 3353 R Lysine>Arginine Non-synonymous SNV
10449 CCC > CAC P 3395 H Proline>Histidine Non-synonymous SNV
11405 GTC > TTC V 3714 F Valine>Phenylalanine Non-synonymous SNV
11285-11293 del3674/3676 del3674/3676 Non-frame shift deletion
11537 ATT > GTT I 3758 V Isoleucine>Valine Non-synonymous SNV

ORF3a (25393…26220)
25471 GAT > TAT D 27 Y Aspartic acid>Tyrosine Non-synonymous SNV
ORF1b (13468...21555)
13756 ATA > GTA I 97 V Isoleucine>Valine Non-synonymous SNV *
14408 CCT > CTT P 314 L Proline>Leucine Non-synonymous SNV *
14821 CCA > TCA P 452 S Proline>Serine Non-synonymous SNV *
15641 AAT > AGT N 725 S Asparagine>Serine Non-synonymous SNV *
15982 GTA > ATA V 839 I Valine>Isoleucine Non-synonymous SNV
16744 GGT > AGT G 1093 S Glycine>Serine Non-synonymous SNV
17410 GGT > TGT R 1315 C Arginine>Cisteine Non-synonymous SNV
18163 ATA > GTA I 1566 V Isoleucine>Valine Non-synonymous SNV
18433 GAT > CAT D 165 H Aspartic acid>Histidine Non-synonymous SNV
19999 GTT > TTT V 2178 F Valine>Phenylalanine Non-synonymous SNV
20003 GAT > GGT P 2179 G Proline>Glycine Non-synonymous SNV
within the SARS-COV-2 genome isolated from Duhok/Iraq

S
(21563...25384) Non-synonymous SNV
21765 - 21770 TACATG > - - - del69/70 del69/70 Non-synonymous SNV
21846 ACT > ATT T95I Threonine>Isoleucine Non-frame shift deletion
21987 GGT > GAT G142D Glycine>Aspartic acid Non-synonymous SNV
21987 - 21995 del142/144 del142/144 Non-frame shift deletion
21996 TAC > -AC Y 145 D Tyrosine>Aspartic acid Non-synonymous SNV
22194 - 22196 AAT > A-- del211/211 del211/211 Non-synonymous SNV
22197 TTA > -TA L 212 I Leucine>Isoleucine Non-synonymous SNV
222000 GTG > GGG V 213 G Valine>Glycine Non-synonymous SNV
22578 GCT > GAT G339D Glycine>Aspartic acid Non-synonymous SNV
22599 AGA > AAA R346K Arginine>Lysine Non-synonymous SNV
22673 T > C S371L Serine>Leucine Non-synonymous SNV
22674 C > T S 373 P Serine>Proline Non-synonymous SNV
22686 TCC > TTC S 375 F Serine>Phenylalanine Non-synonymous SNV
22688 ACT > GCT T 376 A Threonine>Isoleucine Non-synonymous SNV
22786 AGA > AGC R408S Arginine>Serine Non-synonymous SNV
22813 AAG > AAT K 417 N Lysine>Asparagine Non-synonymous SNV *
22882 AAT > AAG N440K Asparagine>Lysine Non-synonymous SNV
22898 GGT > AGT G446S Glycine>Serine Non-synonymous SNV *
23013 GAA > GCA E 484 A Glutamic acid > isoleucine Non-synonymous SNV *
22992 AGC > AAC S477N Serine>Asparagine Non-synonymous SNV *
22995 ACA > AAA T478K Threonine>Lysine Non-synonymous SNV *
23040 CAA > CGA Q493R Glutamine>Arginine Non-synonymous SNV *
23048 G > A G496S Glycine>Serine Non-synonymous SNV *
23055 A > G Q498R Glutamine>Arginine Non-synonymous SNV *
23063 AAT > TAT N501Y Asparagine>Tyrosine Non-synonymous SNV *
23075 TAC > CAC Y505H Tyrosine>Histidine Non-synonymous SNV *
23202 ACA > AAA T547K Threonine>Lysine Non-synonymous SNV
23403 GAT > GGT D614G Aspartic acid>Glycine Non-synonymous SNV
23525 CAT > TAT H655Y Histidine>Tyrosine Non-synonymous SNV
23599 T > G N679K Asparagine>Lysine Non-synonymous SNV
23604 CCT > CAT P681H Proline>Histidine Non-synonymous SNV
23854 AAC > AAA N764K Asparagine>Lysine Non-synonymous SNV
23948 GAT > TAT D796Y Aspartic acid>Tyrosine Non-synonymous SNV
24130 ACC > AAA N856K Asparagine>Lysine Non-synonymous SNV
24424 CAA > CAT Q954H Glutamine>Histidine Non-synonymous SNV
24469 AAT > AAA N969K Asparagine>Lysine Non-synonymous SNV
24503 CCT > TTT L981F Leucine>Phenylalanine Non-synonymous SNV

E (26245... 26472) 26270 ACA > ATA T 9 I Threonine>Isoleucine Non-synonymous SNV
M (26523... 27191) 26530 GAT > GGT D 3 G Aspartic acid>Glycine Non-synonymous SNV
26577 CAA > GAA Q 19 E Glutamine>Glutamic acid Non-synonymous SNV
26709 GCT > ACT A 63 T Alanine>Threonin Non-synonymous SNV **
ORF6 ( 27202…27387) 27269 AAA > -AA K 23 * K23* Non-synonymous SNV
27266 - 27268 TTA > - - - del22/23 del22/23 Non-frame shift deletion
ORF9b (
28284…28577) 28311 CCC > TCC P 10 S Proline>Serine Non-synonymous SNV
N (28274…29533) 28881 AGG > AAA R 203 K Arginine>Lysine Non-synonymous SNV
28882 AGG > AAA R203 K Arginine>Lysine Non-synonymous SNV *
28883 GGA > ACG G 204 R Glycine>Arginine Non-synonymous SNV *
28311 CCC > CTC P 13 L Proline>Leucine Non-synonymous SNV *
28725 CCT > CTT P 151 L Proline>Leucine Non-synonymous SNV *
29000 GGC > AGC G 243 S Glycine>Serine Non-synonymous SNV

List of variations displayed in structure
• A67V H69del V70del(69) T95I G142D V143del Y144del(143) Y145del(143) N211del L212I
ins214EPE R346K S371L S373P S375F K417N N440K G446S S477N T478K E484A Q493R
G496S Q498R N501Y Y505H T547K D614G H655Y N679K(674) P681H(674) N764K D796Y
N856K Q954H N969K L981F

Conclusion
• These findings provide data on the mutation pattern in circulating
variants in
the country, which could help public health authorities in issuing
and updating the control roles for SARS-CoV-2 emergence.
• Here we recommend future studies focus on the
impact and function of such genomic variants on the virus's
infectivity, pathogenesis, and severity.
• In addition developing a new vaccine, especially multivalent
vaccines containing multiple VOCs, could be a good step forward in
controlling the latest infections

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