Observation of SARS-CoV-2 genome characteristics and clinical manifestations within eight family clusters from GH and GK clades in Jakarta, Indonesia

Fera Ibrahim; Augustine Natasha; Andi Yasmon; Fithriyah Fithriyah; Anis Karuniawati; Rivia Gina Rahmawaty; Yulia Saharman; Pratiwi Sudarmono

doi:10.12688/f1000research.122019.1

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Research Article

Observation of SARS-CoV-2 genome characteristics and clinical manifestations within eight family clusters from GH and GK clades in Jakarta, Indonesia

[version 1; peer review: 1 approved with reservations, 1 not approved]

Fera Ibrahim ^1,2, Augustine Natasha^1,3, Andi Yasmon¹, [...] Fithriyah Fithriyah¹, Anis Karuniawati^1,2, Rivia Gina Rahmawaty^1,2, Yulia Saharman^1,2, Pratiwi Sudarmono^1,2

Fera Ibrahim ^1,2, Augustine Natasha^1,3, [...] Andi Yasmon¹, Fithriyah Fithriyah¹, Anis Karuniawati^1,2, Rivia Gina Rahmawaty^1,2, Yulia Saharman^1,2, Pratiwi Sudarmono^1,2

PUBLISHED 02 Aug 2022

Author details Author details

¹ Clinical Microbiology Department, Faculty of Medicine, Universitas Indonesia, Jakarta, Jakarta, Indonesia
² Clinical Microbiology Department, Dr. Cipto Mangunkusumo Hospital, Jakarta, Jakarta, Indonesia
³ Clinical Microbiology, Genomik Solidaritas Indonesia Laboratory, Jakarta, Jakarta, Indonesia

Fera Ibrahim
Roles: Conceptualization, Formal Analysis, Methodology, Supervision, Validation, Writing – Review & Editing

Augustine Natasha
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Project Administration, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Andi Yasmon
Roles: Formal Analysis, Investigation, Methodology, Supervision, Validation, Writing – Review & Editing

Fithriyah Fithriyah
Roles: Data Curation, Investigation, Methodology, Supervision, Writing – Review & Editing

Anis Karuniawati
Roles: Investigation, Supervision, Validation

Rivia Gina Rahmawaty
Roles: Data Curation, Investigation, Methodology

Yulia Saharman
Roles: Conceptualization, Supervision, Writing – Review & Editing

Pratiwi Sudarmono
Roles: Conceptualization, Funding Acquisition, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Genomics and Genetics gateway.

This article is included in the Nanopore Analysis gateway.

This article is included in the Coronavirus (COVID-19) collection.

Abstract

Background: SARS-CoV-2 rapid mutation generates many concerning new strains. Although lockdown had been applied to contain the disease, the household remains a critical place for its transmission. This study aimed to assess the variation of SARS-CoV-2 strains and their clinical manifestations within family clusters in Jakarta, Indonesia.
Method: Naso-oropharyngeal swab specimens from family clusters positive for SARS-CoV-2 were collected for whole-genome sequencing. Their baseline data, symptoms, and source of infection were recorded. The whole-genome data was then analyzed with the bioinformatics program to evaluate the SARS-CoV-2 genome characteristic and submitted to GISAID for strain identification. The phylogenetic tree was built to observe the relationship between virus strain within the family cluster and its clinical manifestation.
Result: This study obtained eight family clusters from twenty-two patients. Half of the cluster's source of infection was a family member who had to work at the office. The infection rate ranged from 37.5% to 100%. The phylogenetic tree showed that the same virus within a cluster could generate various clinical outcomes. Five clusters had one family member with pneumonia, while others had mild to no symptoms. Four breakthrough cases were detected in this study, which were infected by the virus from GH and GK clade.
Conclusion: Our study observed the SARS-CoV-2 transmission to the household setting through the workplace, which might become a common pathway if the pandemic status is removed. Although vaccination is expected to reduce the burden of COVID-19, adequate control measures remain essential, given that breakthrough infections are evident.

Keywords

SARS-Cov-2, Family Cluster, Whole-Genome Sequence, Next Generation Sequencing, Breakthrough infection.

Corresponding author: Fera Ibrahim

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2022 Ibrahim F et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Ibrahim F, Natasha A, Yasmon A et al. Observation of SARS-CoV-2 genome characteristics and clinical manifestations within eight family clusters from GH and GK clades in Jakarta, Indonesia [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2022, 11:886 (https://doi.org/10.12688/f1000research.122019.1) First published: 02 Aug 2022, 11:886 (https://doi.org/10.12688/f1000research.122019.1) Latest published: 16 Oct 2023, 11:886 (https://doi.org/10.12688/f1000research.122019.2)

Introduction

SARS-CoV-2 was first detected in late 2019; however, its rapid mutations have resulted in several dominant strains that threaten the efficacy of current treatment and control protocols. D614G was the first reported mutation to rapidly emerge among the dominant viral strains worldwide.¹ This mutation then increased and generated the emergence of the variants of concern, such as Alpha, Beta, Gamma, and Delta, which alternately became the dominant strain in many countries.¹ These variants had been reported to have higher transmissibility, more severe clinical manifestations, and the ability to potentially escape the immunity conferred by the current vaccines.²

Lockdown and quarantine have been the main actions applied to contain the spread of SARS-CoV-2 in the population. Nonetheless, the household setting has been a critical SARS-CoV-2 transmission pathway.³^,⁴ The rate of secondary transmission among household contacts has varied from 16.6% to 30%.³^,⁵ Household transmission is more challenging to control, given the frequent unavoidable contacts among family members sharing the same home.⁶ In addition, transmission within households has been reported to be increased in families of greater size.⁷ The urban areas of Indonesia are less likely to have a family size of fewer than four individuals.⁸ The basic reproduction number in Jakarta during the early spread of COVID-19 was 1.75,⁹ which has made the household setting one of the vital arenas for COVID-19 transmission in Indonesia.

SARS-CoV-2 surveillance has been implemented in many referral laboratories in Indonesia since the middle of 2020, and it has supported researchers in examining the dynamics of SARS-CoV-2 variants throughout the year. This study was performed to assess the variation of SARS-CoV-2 strains and their clinical manifestations within family clusters in Jakarta, Indonesia.

Methods

Ethics and patient sampling

This study was conducted after obtaining ethical clearance from the Research Ethics Committee of Universitas Indonesia (protocol number 20-05-0516). Patients were informed before providing written consent. All the patients involved in this study agreed to have their medical information and specimens used for analysis.

Specimen collection and processing

We selected naso-oropharyngeal swab specimens from family clusters positive for SARS-CoV-2 for whole-genome sequencing, choosing at least two patients to represent each family cluster. The specimens were stored in -80⁰C from the date of detection to the sequencing process. The 200-μl of specimen extraction was performed according to the manufacturer’s instructions (PureLink™ Viral RNA/DNA Mini Kit, Thermo Fisher Scientific, USA). The real-time reverse transcription polymerase chain reaction (PCR) US Centers for Disease Control and Prevention validation test targeting N1 and N2 was performed according to the manufacturer’s instructions (SensiFAST™, Meridian Bioscience, USA).

The total RNA from positive extraction was then cleaned with DNase I and concentrated (RNA Clean & Concentrator^TM-5, Zymo Research, USA). The libraries were prepared according to the ARTIC multiplex PCR method, using an nCoV-19 V3 panel.¹⁰ The libraries were then processed with Oxford Nanopore’s GridION sequencer, employing MinKNOW version 20.06.9 and MinKNOW core version 4.0.11.¹⁰ High accuracy basecalling was conducted using Guppy version 4.0.11.¹¹ All generated reads were assembled using EPI2ME Labs platform employing ARTIC workflow.¹⁰

Variant analysis and phylogenetic tree construction

The complete genome sequences were uploaded to GISAID for identification of clade, lineage, and mutations. The sequences were further annotated using SnpEff v4.3.¹² The phylogenetic tree was built using Geneious Prime 2021.1.1, using UPGMA as the tree build method and Tamura-Nei as the genetic distance model, with NC_045512 as the reference sequence.

Results

This study included 22 patients from eight family clusters, performed from January 2021 to August 2021. The patient characteristics are shown in Table 1. The phylogenetic tree is shown in Figure 1.

Table 1. Characteristics of the patients included in the study.

Patient	Cluster	Sex	Age range (y.o.)	Dates of detection	Symptoms						Confirmed pneumonia	Severity degree	Vaccination history before infection	Clade	Pango lineage
Patient	Cluster	Sex	Age range (y.o.)	Dates of detection	Fever	Anosmia	Cough	Malaise	Myalgia	Gastrointestinal problem	Confirmed pneumonia	Severity degree	Vaccination history before infection	Clade	Pango lineage
Patient 1	A	Male	20-29	Jan 2021	+		+				No	Mild	No	GH	B.1.459
Patient 2		Female	< 10	Jan 2021		+					No	Mild	No	GH	B.1.459
Patient 3		Male	60-69	Jan 2021	+		+	+		+	Yes	Moderate	No	GH	B.1.459
Patient 4	B	Female	20-29	Jan 2021				+	+	+	No	Mild	No	GH	B.1.
Patient 5		Female	50-59	Jan 2021	+	+		+	+	+	No	Mild	No	GH	B.1.
Patient 6		Female	20-29	Jan 2021	+		+	+	+	+	Yes	Moderate	No	GH	B.1.
Patient 7	C	Male	40-49	Jan 2021	+		+	+		+	Yes	Moderate	No	GH	B.1.466.2
Patient 8		Male	< 10	Jan 2021							No	Asymptomatic	No	GH	B.1.466.2
Patient 9		Male	< 10	Jan 2021							No	Asymptomatic	No	GH	B.1.466.2
Patient 10	D	Female	40-49	Feb 2021			+	+			No	Mild	No	GH	B.1.466.2
Patient 11		Male	40-49	Feb 2021	+		+	+			Yes	Moderate	No	GH	B.1.466.2
Patient 12		Female	10-19	Feb 2021		+		+			No	Mild	No	GH	B.1.466.2
Patient 13	E	Female	50-59	Apr 2021	+		+	+		+	No	Mild	Yes (Sinovac, 2 doses)	GH	B.1.466.2
Patient 14	E	Male	10-19	Apr 2021							No	Asymptomatic	No	GH	B.1.466.2
Patient 15	F	Female	20-29	May 2021	+		+	+		+	No	Mild	Yes (Sinovac, 2 doses)	GH	B.1.1.526
Patient 16	F	Male	60-69	Jun 2021							No	Mild	Yes (Sinovac, 2 doses)	GK	B.1.617.2 + AY24
Patient 17	G	Female	40-49	Jun 2021	+	+	+		+		No	Mild	Yes (Sinovac, 1 doses)	GK	B.1.617.2 + AY23
Patient 18		Female	50-59	Jun 2021	+	+	+		+		No	Mild	No	GK	B.1.617.2 + AY23
Patient 19		Male	50-59	Jun 2021	+	+	+		+		No	Mild	No	GK	B.1.617.2 + AY23
Patient 20	H	Female	30-39	Aug 2021	+		+	+	+	+	Yes	Moderate	Yes (Sinovac, 2 doses)	GK	B.1.617.2 + AY23
Patient 21		Female	< 10	Aug 2021	+					+	No	Mild	No	GK	B.1.617.2 + AY23
Patient 22		Female	50-59	Aug 2021	+		+	+	+	+	No	Mild	No	GK	B.1.617.2 + AY23

Figure 1. Phylogenetic tree of all the clusters.

The source of infection in clusters A and D was unknown, and the infections were detected due to the screening program; the source of infection in clusters B, C, E, and F was a family member who had a history of working from the office during the pandemic; the source of infection in cluster G was a family member who had a history of traveling to another province via airplane for business assignments; and the source of infection in cluster H was the nanny who worked in the house during the day. Five patients had been vaccinated, with four of them completing the second dose.

In clusters C (4 of 4), D (5 of 5), E (5 of 5), F (3 of 3), G (4 of 4), and H (4 of 4), the whole family had become infected with SARS-CoV-2. For cluster A, only three of eight family members became infected with COVID-19. For cluster B, 4 of 5 family members caught COVID-19. The infection rate ranged from 37.5% to 100%.

Nineteen of the 22 patients isolated at home, with patients six, seven, 11, and 20 having pneumonia during their isolation, but who were not able to be admitted to the hospital because of the lack of available beds. However, these patients had access to the outpatient COVID-19 clinics to obtain doctor assessment, symptomatic medications, and radiology tests. The only patient with pneumonia who was able to be admitted to the hospital was patient three. Most patients did not have their blood tested during their early days after being detected positive due to difficulties reaching the healthcare service at the peak of the first and second wave. The Pango lineages circulating within the clusters were B.1.459, B.1., B.1.466.2, B.1.1.526, B.1.617.2 + AY23, and AY24. Most clusters had the same strain within each patient, except cluster F. For cluster F, patient 16 was detected positive after regular screening because he took care of patient 15, which explains the distance between the dates of the SARS-CoV-2 positive results between the patients.

This study had 2 dominant clades, GH and GK. The evaluation of the variation rates, transition-transversion (ti/tv) ratio, total single nucleotide polymorphism (SNP), total deletion, total variation, missense-silent ratio, spike (S) and nucleocapsid (N) gene nonsynonymous mutations, and total nonsynonymous mutation are shown in Table 2.

Table 2. Bioinformatics analysis results between clade GH and GK.

	GH	GK
Variation rate (Median, IQR)	0.00083 (0.0002)	0.0013 (0.0003)
Ti/Tv ratio (Median, IQR)	4 (0.83)	1.8 (0.10)
Transition (Median, IQR)	40 (12)	48 (19)
Transversion (Median, IQR)	10 (6)	18 (9)
Total variant (Median, IQR)	25 (6)	40 (9)
Total SNP (Median, IQR)	24 (6)	37 (9)
Total Deletion (Median, IQR)	1 (1)	3 (0)
Missense-silent Ratio (Median, IQR)	1.11 (0.49)	3.4 (1.72)
Total nonsynonymous mutation (Median, IQR)	16 (5)	32 (4)
Total S mutation (Median, IQR)	3 (1)	11 (1)
Total N mutation (Median, IQR)	2 (1)	4 (1)
Total SNP mutation (Median, IQR)	6 (7)	12 (0)

In addition, given that the spike gene had the highest number of mutations, the distribution of its nonsynonymous mutations within the patients is shown in Table 3.

Table 3. The distribution of the nonsynonymous mutations of the SARS-CoV-2 spike gene.

Patient	Cluster	A222V	D614G	D950N	E156G	F157del	G142D	H49Y	L18F	L452R	N439K	P26L	P681R	R158del	S689R	S494P	T19R	T478K	T716I	V1264L
Patient 1	A		+						+						+				+
Patient 2			+						+						+				+
Patient 3^*			+						+						+				+
Patient 4	B		+					+	+							+
Patient 5			+					+	+							+
Patient 6^*			+					+	+							+
Patient 7^*	C		+								+
Patient 8			+								+
Patient 9			+								+
Patient 10	D		+								+		+
Patient 11^*			+								+		+
Patient 12			+								+		+
Patient 13	E		+								+		+
Patient 14	E		+								+		+
Patient 15	F		+								+		+
Patient 16	F	+	+	+	+	+	+			+			+	+			+	+
Patient 17	G		+	+	+	+	+			+			+	+			+	+		+
Patient 18			+	+	+	+	+			+			+	+			+	+		+
Patient 19			+	+	+	+	+			+			+	+			+	+		+
Patient 20^*	H		+	+	+	+	+			+		+	+	+			+	+		+
Patient 21			+	+	+	+	+			+		+	+	+			+	+		+
Patient 22			+	+	+	+	+			+		+	+	+			+	+		+

* Patients with pneumonia.

Discussion

Four clusters in our study had an index patient with a history of working in an office. Cases of SARS-CoV-2 infection from office workers outside the healthcare setting were scarcely reported.¹³ Six of eight family clusters had a 100% infection rate, showing the efficacy of SARS-CoV-2 transmission within the family group. As reported by several studies, the household infection rate ranged between 75% and 100%.³^,¹⁴ The high infection rate within households combined with poor preventive measures in the workplace could generate a spike in COVID-19 cases.

This study also found SARS-CoV-2 infections in patients who had been vaccinated (breakthrough cases). Infection after complete vaccination was possible, no matter which type of vaccine had been administered.¹⁵^,¹⁶ Duarte et al. reported the breakthrough cases mainly were mild, and that other factors might have influenced the symptomatic infections, given that they were not correlated with lack of vaccine-induced immunity.¹⁵ Although our study could not represent the general population, the concern of vaccine efficacy in the face of new variants with more mutations has become relevant since Christensen et al. reported an increase in breakthrough cases caused by the Delta variant (as part of the GK clade).¹⁷ Therefore, preventive measures such as wearing a mask are still necessary, even after full vaccination.

The clinical manifestation varied within the patients in each family cluster. This situation was in concordance with several case reports, which have mentioned variations in disease severity within a family cluster.¹⁸ However, seven clusters were monophyletic, which showed that the same virus could generate a different outcome. Furthermore, we found the virus genome of the patients with pneumonia was similar to the other patients without pneumonia (Table 1, Figure 1). No specific variation or combination in the genome mutation could generate a particular clinical outcome (Table 3). The D614G spike mutation was also distributed evenly within the patients. This finding was in concordance with Korber et al. and Lorenzo-Redondo et al., who noted that the D614G mutations did not correlate with more severe outcomes.¹⁹^,²⁰

Our study showed a ti/tv ratio decline between the GH and GK clades. This decline was in concordance with the study by Duchêne et al., which revealed the ti/tv scaled negatively over time, especially for rapidly evolving RNA viruses.²¹ We also found an increase in the GK total spike gene mutations compared with the GH clade. Given that the GK clade appeared later in our study, it was expected to have more mutations because the RNA virus would utilize various mechanisms of genetic variation to guarantee its survival.²² The evolving behavior can facilitate the virus to evade the current vaccine-generated immunity, resulting in breakthrough infections.

One limitation of our study was the small number of patients and family clusters. This study could not provide serological test results, especially from patients with vaccination history, given that there were no guidelines to confirm an immune response after vaccination. Our study showed that the same virus within a cluster could generate various clinical outcomes. Our observation showed the SARS-CoV-2 transmission in the household setting through the workplace, which could be a common pathway in the future of COVID-19 once the pandemic status is lifted. Although vaccination is expected to reduce the burden of COVID-19, control measures are crucial, given that breakthrough infections due to a new variant are evident.

Data availability

The virus genome included in this study was published at GISAID with accession ID:

EPI_ISL_2692986, EPI_ISL_2692987, EPI_ISL_2692989, EPI_ISL_2993633, EPI_ISL_2993659, EPI_ISL_2993632, EPI_ISL_2692990, EPI_ISL_2692991, EPI_ISL_2692992, EPI_ISL_2993434, EPI_ISL_2993539, EPI_ISL_2993658, EPI_ISL_2692909, EPI_ISL_2692981, EPI_ISL_2692983, EPI_ISL_3000155, EPI_ISL_3456060, EPI_ISL_4847411, EPI_ISL_4847413, EPI_ISL_5054899, EPI_ISL_5054915, EPI_ISL_5054918.

Acknowledgements

The authors would like to thank Universitas Indonesia, through PUTI Grant with contract number NKB-1493/UN2.RST/HKP.05.00.2020.

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Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 02 Aug 2022

Author details Author details

Fera Ibrahim
Roles: Conceptualization, Formal Analysis, Methodology, Supervision, Validation, Writing – Review & Editing

Andi Yasmon
Roles: Formal Analysis, Investigation, Methodology, Supervision, Validation, Writing – Review & Editing

Fithriyah Fithriyah
Roles: Data Curation, Investigation, Methodology, Supervision, Writing – Review & Editing

Anis Karuniawati
Roles: Investigation, Supervision, Validation

Rivia Gina Rahmawaty
Roles: Data Curation, Investigation, Methodology

Yulia Saharman
Roles: Conceptualization, Supervision, Writing – Review & Editing

Pratiwi Sudarmono
Roles: Conceptualization, Funding Acquisition, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (2)

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https://doi.org/10.12688/f1000research.122019.2

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https://doi.org/10.12688/f1000research.122019.1

© 2022 Ibrahim F et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Ibrahim F, Natasha A, Yasmon A et al. Observation of SARS-CoV-2 genome characteristics and clinical manifestations within eight family clusters from GH and GK clades in Jakarta, Indonesia [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2022, 11:886 (https://doi.org/10.12688/f1000research.122019.1)

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Reviewer Report 23 Aug 2022

Serafeim C. Chaintoutis, Diagnostic Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece

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https://doi.org/10.5256/f1000research.133953.r146400

The article "Observation of SARS-CoV-2 genome characteristics and clinical manifestations within eight family clusters from GH and GK clades in Jakarta, Indonesia" by F. Ibrahim et al., provides basically the genetic findings (based on NGS analysis) on the genetic characteristics of SARS-CoV-2 strains retrieved from 8 infected families (“family clusters”) in Jakarta as of Jan. 2021. In my opinion, the present manuscript has only geographically limited interest and brings limited novel findings to the international scientific community.
Main concern
I am concerned about whether the findings are representative of the situation in Jakarta, at the time of sampling, as there is no mention of how the 8 families that were included in the analysis were selected, geographical characteristics, and so on. The authors should present how those family clusters were selected and investigated.

Specific comments:

Abstract:

"Although lockdown had been applied to contain the disease, the household remains a critical place for its transmission." The two parts of the sentence are not combinable in a scientific manner. Several measures were applied to contain the disease. And many places remain critical for transmission. This also applies to the introduction.
"Naso-oropharyngeal swab..." Do authors mean both nasopharyngeal and oropharyngeal? Please elaborate.
"The infection rate ranged from 37.5 to 100%". You mean within families?
"Four breakthrough cases were detected..." Do authors mean vaccine breakthrough?

Introduction:

"D614G was the first reported mutation..." This is technically an amino-acid substitution. Please, be precise.
"This mutation then increased and generated the emergence of the variants of concern". How is this supported? This is scientifically unjustified.
"This study was performed to assess the variation of SARS-CoV-2 strains and their clinical manifestations within family clusters in Jakarta, Indonesia". By this, the impression that of large-scale study is being given. However, 8 families were only investigated. This is not representative. How were these family clusters chosen? Was there any reason? There might be a bias in the selection of families/research material, with potential false community representation results.

Materials and Methods:

Details on the sequencing methodology and analysis are missing. Herein, sequencing was performed using "Oxford Nanopore’s GridION sequencer", with read depth lower than Illumina or Ion Torrent systems usings critical to verify that the identified mutations are not an "artifact" of the sequencing process.

References:

Need to be checked and verified.

Fig. 1:

The relevant caption is missing several important pieces of information. What type of tree is this? Many trees are characterized as "phylogenetic trees" Details are needed on how this analysis was performed and the tree was constructed. This information needs to be added to the figure caption. The information provided on the branches is not easy to read and understand.

Tables:

Table 1. This table presents Pango lineages (i.e., B.1) that were not anticipated as of January 2021.
Table 2. It is not clear whether the authors mean in fact mutations, or substitutions by "total N mutations", "total S mutations", etc.
Table 3. This table also presents amino-acid substitutions, but they are named "mutations". Authors need to be precise.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Clinical microbiology, infectious diseases, immunology, virology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

Report a concern

Author Response 16 Oct 2023

Fera Ibrahim, Clinical Microbiology Department, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

16 Oct 2023

Author Response
Dear Serafeim C. Chaintoutis,

Thank you very much for your time and effort to review our study.

Here's our response for each input:

Abstract:
1. "Although
... Continue reading
Dear Serafeim C. Chaintoutis,

Thank you very much for your time and effort to review our study.

Here's our response for each input:

Abstract:

"Although lockdown had been applied to contain the disease, the household remains a critical place for its transmission." The two parts of the sentence are not combinable in a scientific manner. Several measures were applied to contain the disease. And many places remain critical for transmission. This also applies to the introduction.

Response: Revised sentence: The lockdown had been applied to prevent transmission of virus, even the transmission in household remains a critical problem especially in poor and developing countries with more burden family.

"Naso-oropharyngeal swab..." Do authors mean both nasopharyngeal and oropharyngeal? Please elaborate.

Response: yes, it refer to nasopharyngeal and oropharyngeal.

"The infection rate ranged from 37.5 to 100%". You mean within families?

Response: yes, this rate resembles the infection rate in the families. Revised sentence: Therefore, the infection rate in family ranged from 37.5% to 100%.

"Four breakthrough cases were detected..." Do authors mean vaccine breakthrough?

Response: it means COVID19 occur even after the individual received a vaccine. Revised sentence: Four vaccine breakthrough cases were detected in this study, which were infected by the virus from GH and GK clade.

Introduction:

"D614G was the first reported mutation..." This is technically an amino-acid substitution. Please, be precise.

Response: it is an amino-acid substitution, thank you very much for the corrective comment; we will change this “mutation” in to “amino acid substitution” on whole manuscript.

"This mutation then increased and generated the emergence of the variants of concern". How is this supported? This is scientifically unjustified.

Response: we agree that mutation not always drive to the increased or generated novel voc, thus this sentence will be change in to “The mutation may increase and generate the emergence of the variants of concern”

"This study was performed to assess the variation of SARS-CoV-2 strains and their clinical manifestations within family clusters in Jakarta, Indonesia". By this, the impression that of large-scale study is being given. However, 8 families were only investigated. This is not representative. How were these family clusters chosen? Was there any reason? There might be a bias in the selection of families/research material, with potential false community representation results.

Response: samples on this study are from government program of COVID-19 tracking which RT-PCR diagnosis only taken place when symptoms are appears, thus it was limitations in this study.

Materials and Methods:

Details on the sequencing methodology and analysis are missing. Herein, sequencing was performed using "Oxford Nanopore’s GridION sequencer", with read depth lower than Illumina or Ion Torrent systems usings critical to verify that the identified mutations are not an "artifact" of the sequencing process.[P1]

Response: The methods for nanopore sequencing were done exactly as the protocols described in the citations (Citations number 10 and 11) and according to manufacturers instructions. For the depth, as manufacturers suggestions and based on the citations, the sequencing was set to achieve 100x depth of coverage across all targets. Therefore, all the sequencing results only can be proceeded for further analysis if the 100x depth of coverage was achieved. We determined the sequence was good enough for further analysis because we used more depth than required (50x) and run only 10 specimens per nanopore’s flowcells to achieve our desired coverage. We will add this information to the manuscript for the confirmation. In addition, sequencing using illumina platform was not feasible in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives.

Fig. 1:

The relevant caption is missing several important pieces of information. What type of tree is this? Many trees are characterized as "phylogenetic trees" Details are needed on how this analysis was performed and the tree was constructed. This information needs to be added to the figure caption. The information provided on the branches is not easy to read and understand.[P2]

Response: The phylogenetic tree was built using Geneious Prime 2021.1.1, using UPGMA as the tree build method and Tamura-Nei as the genetic distance model, with NC_045512 as the reference sequence. We had mentioned it into our manuscript.

Tables:

Table 1. This table presents Pango lineages (i.e., B.1) that were not anticipated as of January 2021.

Table 2. It is not clear whether the authors mean in fact mutations, or substitutions by "total N mutations", "total S mutations", etc.

Response: They refer to amino acid substitution within N or S protein.

Table 3. This table also presents amino-acid substitutions, but they are named "mutations". Authors need to be precise.

Response: Thank you very much for the corrective comments, the data presented in Table 3 are refer to amino acid substitutions, thus we will revise its “mutation” in to “amino acid substitution”.
Dear Serafeim C. Chaintoutis,

Thank you very much for your time and effort to review our study.

Here's our response for each input:

Abstract:

"Although lockdown had been applied to contain the disease, the household remains a critical place for its transmission." The two parts of the sentence are not combinable in a scientific manner. Several measures were applied to contain the disease. And many places remain critical for transmission. This also applies to the introduction.

Response: Revised sentence: The lockdown had been applied to prevent transmission of virus, even the transmission in household remains a critical problem especially in poor and developing countries with more burden family.

"Naso-oropharyngeal swab..." Do authors mean both nasopharyngeal and oropharyngeal? Please elaborate.

Response: yes, it refer to nasopharyngeal and oropharyngeal.

"The infection rate ranged from 37.5 to 100%". You mean within families?

Response: yes, this rate resembles the infection rate in the families. Revised sentence: Therefore, the infection rate in family ranged from 37.5% to 100%.

"Four breakthrough cases were detected..." Do authors mean vaccine breakthrough?

Response: it means COVID19 occur even after the individual received a vaccine. Revised sentence: Four vaccine breakthrough cases were detected in this study, which were infected by the virus from GH and GK clade.

Introduction:

"D614G was the first reported mutation..." This is technically an amino-acid substitution. Please, be precise.

Response: it is an amino-acid substitution, thank you very much for the corrective comment; we will change this “mutation” in to “amino acid substitution” on whole manuscript.

"This mutation then increased and generated the emergence of the variants of concern". How is this supported? This is scientifically unjustified.

Response: we agree that mutation not always drive to the increased or generated novel voc, thus this sentence will be change in to “The mutation may increase and generate the emergence of the variants of concern”

"This study was performed to assess the variation of SARS-CoV-2 strains and their clinical manifestations within family clusters in Jakarta, Indonesia". By this, the impression that of large-scale study is being given. However, 8 families were only investigated. This is not representative. How were these family clusters chosen? Was there any reason? There might be a bias in the selection of families/research material, with potential false community representation results.

Response: samples on this study are from government program of COVID-19 tracking which RT-PCR diagnosis only taken place when symptoms are appears, thus it was limitations in this study.

Materials and Methods:

Details on the sequencing methodology and analysis are missing. Herein, sequencing was performed using "Oxford Nanopore’s GridION sequencer", with read depth lower than Illumina or Ion Torrent systems usings critical to verify that the identified mutations are not an "artifact" of the sequencing process.[P1]

Response: The methods for nanopore sequencing were done exactly as the protocols described in the citations (Citations number 10 and 11) and according to manufacturers instructions. For the depth, as manufacturers suggestions and based on the citations, the sequencing was set to achieve 100x depth of coverage across all targets. Therefore, all the sequencing results only can be proceeded for further analysis if the 100x depth of coverage was achieved. We determined the sequence was good enough for further analysis because we used more depth than required (50x) and run only 10 specimens per nanopore’s flowcells to achieve our desired coverage. We will add this information to the manuscript for the confirmation. In addition, sequencing using illumina platform was not feasible in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives.

Fig. 1:

The relevant caption is missing several important pieces of information. What type of tree is this? Many trees are characterized as "phylogenetic trees" Details are needed on how this analysis was performed and the tree was constructed. This information needs to be added to the figure caption. The information provided on the branches is not easy to read and understand.[P2]

Response: The phylogenetic tree was built using Geneious Prime 2021.1.1, using UPGMA as the tree build method and Tamura-Nei as the genetic distance model, with NC_045512 as the reference sequence. We had mentioned it into our manuscript.

Tables:

Table 1. This table presents Pango lineages (i.e., B.1) that were not anticipated as of January 2021.

Table 2. It is not clear whether the authors mean in fact mutations, or substitutions by "total N mutations", "total S mutations", etc.

Response: They refer to amino acid substitution within N or S protein.

Table 3. This table also presents amino-acid substitutions, but they are named "mutations". Authors need to be precise.

Response: Thank you very much for the corrective comments, the data presented in Table 3 are refer to amino acid substitutions, thus we will revise its “mutation” in to “amino acid substitution”.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 16 Oct 2023

Fera Ibrahim, Clinical Microbiology Department, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

16 Oct 2023

Author Response
Dear Serafeim C. Chaintoutis,

Thank you very much for your time and effort to review our study.

Here's our response for each input:

Abstract:
1. "Although
... Continue reading
Dear Serafeim C. Chaintoutis,

Thank you very much for your time and effort to review our study.

Here's our response for each input:

Abstract:

"Although lockdown had been applied to contain the disease, the household remains a critical place for its transmission." The two parts of the sentence are not combinable in a scientific manner. Several measures were applied to contain the disease. And many places remain critical for transmission. This also applies to the introduction.

Response: Revised sentence: The lockdown had been applied to prevent transmission of virus, even the transmission in household remains a critical problem especially in poor and developing countries with more burden family.

"Naso-oropharyngeal swab..." Do authors mean both nasopharyngeal and oropharyngeal? Please elaborate.

Response: yes, it refer to nasopharyngeal and oropharyngeal.

"The infection rate ranged from 37.5 to 100%". You mean within families?

Response: yes, this rate resembles the infection rate in the families. Revised sentence: Therefore, the infection rate in family ranged from 37.5% to 100%.

"Four breakthrough cases were detected..." Do authors mean vaccine breakthrough?

Response: it means COVID19 occur even after the individual received a vaccine. Revised sentence: Four vaccine breakthrough cases were detected in this study, which were infected by the virus from GH and GK clade.

Introduction:

"D614G was the first reported mutation..." This is technically an amino-acid substitution. Please, be precise.

Response: it is an amino-acid substitution, thank you very much for the corrective comment; we will change this “mutation” in to “amino acid substitution” on whole manuscript.

"This mutation then increased and generated the emergence of the variants of concern". How is this supported? This is scientifically unjustified.

Response: we agree that mutation not always drive to the increased or generated novel voc, thus this sentence will be change in to “The mutation may increase and generate the emergence of the variants of concern”

"This study was performed to assess the variation of SARS-CoV-2 strains and their clinical manifestations within family clusters in Jakarta, Indonesia". By this, the impression that of large-scale study is being given. However, 8 families were only investigated. This is not representative. How were these family clusters chosen? Was there any reason? There might be a bias in the selection of families/research material, with potential false community representation results.

Response: samples on this study are from government program of COVID-19 tracking which RT-PCR diagnosis only taken place when symptoms are appears, thus it was limitations in this study.

Materials and Methods:

Details on the sequencing methodology and analysis are missing. Herein, sequencing was performed using "Oxford Nanopore’s GridION sequencer", with read depth lower than Illumina or Ion Torrent systems usings critical to verify that the identified mutations are not an "artifact" of the sequencing process.[P1]

Response: The methods for nanopore sequencing were done exactly as the protocols described in the citations (Citations number 10 and 11) and according to manufacturers instructions. For the depth, as manufacturers suggestions and based on the citations, the sequencing was set to achieve 100x depth of coverage across all targets. Therefore, all the sequencing results only can be proceeded for further analysis if the 100x depth of coverage was achieved. We determined the sequence was good enough for further analysis because we used more depth than required (50x) and run only 10 specimens per nanopore’s flowcells to achieve our desired coverage. We will add this information to the manuscript for the confirmation. In addition, sequencing using illumina platform was not feasible in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives.

Fig. 1:

The relevant caption is missing several important pieces of information. What type of tree is this? Many trees are characterized as "phylogenetic trees" Details are needed on how this analysis was performed and the tree was constructed. This information needs to be added to the figure caption. The information provided on the branches is not easy to read and understand.[P2]

Response: The phylogenetic tree was built using Geneious Prime 2021.1.1, using UPGMA as the tree build method and Tamura-Nei as the genetic distance model, with NC_045512 as the reference sequence. We had mentioned it into our manuscript.

Tables:

Table 1. This table presents Pango lineages (i.e., B.1) that were not anticipated as of January 2021.

Table 2. It is not clear whether the authors mean in fact mutations, or substitutions by "total N mutations", "total S mutations", etc.

Response: They refer to amino acid substitution within N or S protein.

Table 3. This table also presents amino-acid substitutions, but they are named "mutations". Authors need to be precise.

Response: Thank you very much for the corrective comments, the data presented in Table 3 are refer to amino acid substitutions, thus we will revise its “mutation” in to “amino acid substitution”.
Dear Serafeim C. Chaintoutis,

Thank you very much for your time and effort to review our study.

Here's our response for each input:

Abstract:

"Although lockdown had been applied to contain the disease, the household remains a critical place for its transmission." The two parts of the sentence are not combinable in a scientific manner. Several measures were applied to contain the disease. And many places remain critical for transmission. This also applies to the introduction.

Response: Revised sentence: The lockdown had been applied to prevent transmission of virus, even the transmission in household remains a critical problem especially in poor and developing countries with more burden family.

"Naso-oropharyngeal swab..." Do authors mean both nasopharyngeal and oropharyngeal? Please elaborate.

Response: yes, it refer to nasopharyngeal and oropharyngeal.

"The infection rate ranged from 37.5 to 100%". You mean within families?

Response: yes, this rate resembles the infection rate in the families. Revised sentence: Therefore, the infection rate in family ranged from 37.5% to 100%.

"Four breakthrough cases were detected..." Do authors mean vaccine breakthrough?

Response: it means COVID19 occur even after the individual received a vaccine. Revised sentence: Four vaccine breakthrough cases were detected in this study, which were infected by the virus from GH and GK clade.

Introduction:

"D614G was the first reported mutation..." This is technically an amino-acid substitution. Please, be precise.

Response: it is an amino-acid substitution, thank you very much for the corrective comment; we will change this “mutation” in to “amino acid substitution” on whole manuscript.

"This mutation then increased and generated the emergence of the variants of concern". How is this supported? This is scientifically unjustified.

Response: we agree that mutation not always drive to the increased or generated novel voc, thus this sentence will be change in to “The mutation may increase and generate the emergence of the variants of concern”

"This study was performed to assess the variation of SARS-CoV-2 strains and their clinical manifestations within family clusters in Jakarta, Indonesia". By this, the impression that of large-scale study is being given. However, 8 families were only investigated. This is not representative. How were these family clusters chosen? Was there any reason? There might be a bias in the selection of families/research material, with potential false community representation results.

Response: samples on this study are from government program of COVID-19 tracking which RT-PCR diagnosis only taken place when symptoms are appears, thus it was limitations in this study.

Materials and Methods:

Details on the sequencing methodology and analysis are missing. Herein, sequencing was performed using "Oxford Nanopore’s GridION sequencer", with read depth lower than Illumina or Ion Torrent systems usings critical to verify that the identified mutations are not an "artifact" of the sequencing process.[P1]

Response: The methods for nanopore sequencing were done exactly as the protocols described in the citations (Citations number 10 and 11) and according to manufacturers instructions. For the depth, as manufacturers suggestions and based on the citations, the sequencing was set to achieve 100x depth of coverage across all targets. Therefore, all the sequencing results only can be proceeded for further analysis if the 100x depth of coverage was achieved. We determined the sequence was good enough for further analysis because we used more depth than required (50x) and run only 10 specimens per nanopore’s flowcells to achieve our desired coverage. We will add this information to the manuscript for the confirmation. In addition, sequencing using illumina platform was not feasible in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives.

Fig. 1:

The relevant caption is missing several important pieces of information. What type of tree is this? Many trees are characterized as "phylogenetic trees" Details are needed on how this analysis was performed and the tree was constructed. This information needs to be added to the figure caption. The information provided on the branches is not easy to read and understand.[P2]

Response: The phylogenetic tree was built using Geneious Prime 2021.1.1, using UPGMA as the tree build method and Tamura-Nei as the genetic distance model, with NC_045512 as the reference sequence. We had mentioned it into our manuscript.

Tables:

Table 1. This table presents Pango lineages (i.e., B.1) that were not anticipated as of January 2021.

Table 2. It is not clear whether the authors mean in fact mutations, or substitutions by "total N mutations", "total S mutations", etc.

Response: They refer to amino acid substitution within N or S protein.

Table 3. This table also presents amino-acid substitutions, but they are named "mutations". Authors need to be precise.

Response: Thank you very much for the corrective comments, the data presented in Table 3 are refer to amino acid substitutions, thus we will revise its “mutation” in to “amino acid substitution”.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Views

Reviewer Report 15 Aug 2022

So Nakagawa, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan

Approved with Reservations

https://doi.org/10.5256/f1000research.133953.r146395

Ibrahim et al. sequenced SARS-CoV-2 genomes that were obtained from 22 COVID-19 patients in 8 family clusters. Except for one family, the results of SARS-CoV-2 genome analysis strongly suggested that the disease was transmitted by household infection.

The study itself is small but solid, and their observation is still reasonable for the current infection dynamics of COVID-19.

Major comments
The details of each nanopore sequencing should be shown in the manuscript. For nanopore sequencing, the quality is known to be relatively low compared to Illumina sequencing. Therefore, if the depth is insufficient, it is impossible to distinguish whether those are mutations or sequencing errors. Please provide more information on the sequencing status (including depth, coverage, and any “shallow” regions if they exist) for each sample.

Minor comments

Introduction: “D614G” should be clearly explained when it first appeared.
Table 1: Are PANGO IDs isolated for Patients 4 to 6 “B.1”? The B.1 lineage is not common in January 2021 in the world. Please verify it.
Figure 1: The legend of the figure is missing, and I could not understand the meaning of the values shown for each branch. In addition, clades and PANGO IDs should be shown, which could be informative for readers.
Table 3: “Total S mutation” and “Total N mutation” are ambiguous. Do these include synonymous mutations as well?
References: Citations should be checked. For example, Reference #1 is not suitable for mentioning SARS-CoV-2 VOCs.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Genomics.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

Report a concern

Author Response 16 Oct 2023

Fera Ibrahim, Clinical Microbiology Department, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

16 Oct 2023

Author Response
Dear So Nakagawa,

Thank you very much for your input and effort to review our paper.

Major comments

The details of each nanopore sequencing should be shown ... Continue reading
Dear So Nakagawa,

Thank you very much for your input and effort to review our paper.

Major comments

The details of each nanopore sequencing should be shown in the manuscript. For nanopore sequencing, the quality is known to be relatively low compared to Illumina sequencing. Therefore, if the depth is insufficient, it is impossible to distinguish whether those are mutations or sequencing errors. Please provide more information on the sequencing status (including depth, coverage, and any “shallow” regions if they exist) for each sample.[P1]

Response: Thank you very much for the inputs. We already use the cutoff 100x depth of coverage for all regions before we proceed for analysis. That’s also why we only included this small number of specimens, since only these specimens could pass such cutoff. In addition, sequencing using illumina platform was not feasible and too expensive in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives. Your message on Illumina is well noted, we will try them if we got more fundings in the future.

Minor comments

Introduction: “D614G” should be clearly explained when it first appeared.

Response: thank you for the input, it’s been revised.

Table 1: Are PANGO IDs isolated for Patients 4 to 6 “B.1”? The B.1 lineage is not common in January 2021 in the world. Please verify it.

Response : According to pango lineage page (Cov-Lineages, B.1 earliest date of detection was January 1^st,2020. Therefore, we’re sure that the B.1 lineage had arrived to Indonesia in 2021. We will add O’Toole’s study as part our methods in terms of evaluating the pangolins lineage.

Figure 1: The legend of the figure is missing, and I could not understand the meaning of the values shown for each branch. In addition, clades and PANGO IDs should be shown, which could be informative for readers.

Response : Thankyou for the input, the figure is revised.

Table 3: “Total S mutation” and “Total N mutation” are ambiguous. Do these include synonymous mutations as well?

Response : the mutations included only the non-synonymous one, as resulted from the GISAID analysis reports.

References: Citations should be checked. For example, Reference #1 is not suitable for mentioning SARS-CoV-2 VOCs.

Response: Thank you for the concerns. We consider your input and we had replaced them with different citations.
Dear So Nakagawa,

Thank you very much for your input and effort to review our paper.

Major comments

The details of each nanopore sequencing should be shown in the manuscript. For nanopore sequencing, the quality is known to be relatively low compared to Illumina sequencing. Therefore, if the depth is insufficient, it is impossible to distinguish whether those are mutations or sequencing errors. Please provide more information on the sequencing status (including depth, coverage, and any “shallow” regions if they exist) for each sample.[P1]

Response: Thank you very much for the inputs. We already use the cutoff 100x depth of coverage for all regions before we proceed for analysis. That’s also why we only included this small number of specimens, since only these specimens could pass such cutoff. In addition, sequencing using illumina platform was not feasible and too expensive in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives. Your message on Illumina is well noted, we will try them if we got more fundings in the future.

Minor comments

Introduction: “D614G” should be clearly explained when it first appeared.

Response: thank you for the input, it’s been revised.

Table 1: Are PANGO IDs isolated for Patients 4 to 6 “B.1”? The B.1 lineage is not common in January 2021 in the world. Please verify it.

Response : According to pango lineage page (Cov-Lineages, B.1 earliest date of detection was January 1^st,2020. Therefore, we’re sure that the B.1 lineage had arrived to Indonesia in 2021. We will add O’Toole’s study as part our methods in terms of evaluating the pangolins lineage.

Figure 1: The legend of the figure is missing, and I could not understand the meaning of the values shown for each branch. In addition, clades and PANGO IDs should be shown, which could be informative for readers.

Response : Thankyou for the input, the figure is revised.

Table 3: “Total S mutation” and “Total N mutation” are ambiguous. Do these include synonymous mutations as well?

Response : the mutations included only the non-synonymous one, as resulted from the GISAID analysis reports.

References: Citations should be checked. For example, Reference #1 is not suitable for mentioning SARS-CoV-2 VOCs.

Response: Thank you for the concerns. We consider your input and we had replaced them with different citations.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 16 Oct 2023

Fera Ibrahim, Clinical Microbiology Department, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

16 Oct 2023

Author Response
Dear So Nakagawa,

Thank you very much for your input and effort to review our paper.

Major comments

The details of each nanopore sequencing should be shown ... Continue reading
Dear So Nakagawa,

Thank you very much for your input and effort to review our paper.

Major comments

The details of each nanopore sequencing should be shown in the manuscript. For nanopore sequencing, the quality is known to be relatively low compared to Illumina sequencing. Therefore, if the depth is insufficient, it is impossible to distinguish whether those are mutations or sequencing errors. Please provide more information on the sequencing status (including depth, coverage, and any “shallow” regions if they exist) for each sample.[P1]

Response: Thank you very much for the inputs. We already use the cutoff 100x depth of coverage for all regions before we proceed for analysis. That’s also why we only included this small number of specimens, since only these specimens could pass such cutoff. In addition, sequencing using illumina platform was not feasible and too expensive in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives. Your message on Illumina is well noted, we will try them if we got more fundings in the future.

Minor comments

Introduction: “D614G” should be clearly explained when it first appeared.

Response: thank you for the input, it’s been revised.

Table 1: Are PANGO IDs isolated for Patients 4 to 6 “B.1”? The B.1 lineage is not common in January 2021 in the world. Please verify it.

Response : According to pango lineage page (Cov-Lineages, B.1 earliest date of detection was January 1^st,2020. Therefore, we’re sure that the B.1 lineage had arrived to Indonesia in 2021. We will add O’Toole’s study as part our methods in terms of evaluating the pangolins lineage.

Figure 1: The legend of the figure is missing, and I could not understand the meaning of the values shown for each branch. In addition, clades and PANGO IDs should be shown, which could be informative for readers.

Response : Thankyou for the input, the figure is revised.

Table 3: “Total S mutation” and “Total N mutation” are ambiguous. Do these include synonymous mutations as well?

Response : the mutations included only the non-synonymous one, as resulted from the GISAID analysis reports.

References: Citations should be checked. For example, Reference #1 is not suitable for mentioning SARS-CoV-2 VOCs.

Response: Thank you for the concerns. We consider your input and we had replaced them with different citations.
Dear So Nakagawa,

Thank you very much for your input and effort to review our paper.

Major comments

The details of each nanopore sequencing should be shown in the manuscript. For nanopore sequencing, the quality is known to be relatively low compared to Illumina sequencing. Therefore, if the depth is insufficient, it is impossible to distinguish whether those are mutations or sequencing errors. Please provide more information on the sequencing status (including depth, coverage, and any “shallow” regions if they exist) for each sample.[P1]

Response: Thank you very much for the inputs. We already use the cutoff 100x depth of coverage for all regions before we proceed for analysis. That’s also why we only included this small number of specimens, since only these specimens could pass such cutoff. In addition, sequencing using illumina platform was not feasible and too expensive in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives. Your message on Illumina is well noted, we will try them if we got more fundings in the future.

Minor comments

Introduction: “D614G” should be clearly explained when it first appeared.

Response: thank you for the input, it’s been revised.

Table 1: Are PANGO IDs isolated for Patients 4 to 6 “B.1”? The B.1 lineage is not common in January 2021 in the world. Please verify it.

Response : According to pango lineage page (Cov-Lineages, B.1 earliest date of detection was January 1^st,2020. Therefore, we’re sure that the B.1 lineage had arrived to Indonesia in 2021. We will add O’Toole’s study as part our methods in terms of evaluating the pangolins lineage.

Figure 1: The legend of the figure is missing, and I could not understand the meaning of the values shown for each branch. In addition, clades and PANGO IDs should be shown, which could be informative for readers.

Response : Thankyou for the input, the figure is revised.

Table 3: “Total S mutation” and “Total N mutation” are ambiguous. Do these include synonymous mutations as well?

Response : the mutations included only the non-synonymous one, as resulted from the GISAID analysis reports.

References: Citations should be checked. For example, Reference #1 is not suitable for mentioning SARS-CoV-2 VOCs.

Response: Thank you for the concerns. We consider your input and we had replaced them with different citations.
Competing Interests: No competing interests were disclosed. Close
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So Nakagawa, Tokai University School of Medicine, Isehara, Japan
Serafeim C. Chaintoutis, Aristotle University of Thessaloniki, Thessaloniki, Greece
Pakorn Aiewsakun, Mahidol University, Bangkok, Thailand
Gusti N Mahardika, Udayana University, Jl. Sesetan-Markisa 6A, Denpasar 80223, Bali,, Indonesia

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26 Jul 2024 | for Version 2

Gusti N Mahardika, Udayana University, Jl. Sesetan-Markisa 6A, Denpasar 80223, Bali,, Indonesia

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Approved With Reservations

This paper is presenting Covid-19 metadata of many clusters which is very few available in the literature, therefore it can be indexed for global audience after major revisions

Some suggested revision were as the following
Specific comments

The phrase/sentences “During the time of the study, the Illumina platform was not feasible and Nanopore platform became the supporter for many small study surveillance groups” is not needed as the paper is not comparing the methods
The accession number NC_045512 should be given more complete information of original standard strains of Wuhan-Hu-1
On the data availability, please refer to term of use of GISAID data by generating epi data set
Please explain the “ti/tv ratio” at the first mention
How to calculate “The infection rate” is not explained. A table explaining the infection rate in specific cluster might display better.
There is an ambiguity in the statement “This study had 2 dominant clades, GH and GK”. Were there other clades? The English expression needs corrected.
Different of equal Clinical manifestation of each clade as well as with or without vaccination is not explained
The author were choosing at least two patients to represent each family cluster for whole genome sequencing, the number of data but the refers In clusters C (4 of 4), D (5 of 5), E (5 of 5), F (3 of 3), G (4 of 4), and H (4 of 4), the whole family had become infected with SARS-CoV-2. For cluster A, only three of eight family members became infected with COVID-19. For cluster B, 4 of 5 family members caught COVID-19.
There is an ambiguity in the discussion statement “Four clusters in our study had an index patient with a history of working in an office”…first this should be in the result. Second, which was the index case is not pinpointing. Third, what office.
“This study also found SARS-CoV-2 infections in patients who had been vaccinated (breakthrough cases)” should be polished like “a vaccine breakthrough phenomenon was observed in this study” or alike
The result is hard to follow. Nineteen of the 22 patients isolated at home, with patients six, seven, 11, and 20 having pneumonia during their isolation, but who were not able to be admitted to the hospital because of the lack of available beds. However, these patients had access to the outpatient COVID-19 clinics to obtain doctor assessment, symptomatic medications, and radiology tests. The only patient with pneumonia who was able to be admitted to the hospital was patient three. Most patients did not have their blood tested during their early days after being detected positive due to difficulties reaching the healthcare service at the peak of the first and second wave. The Pango lineages circulating within the clusters were B.1.459, B.1., B.1.466.2, B.1.1.526, B.1.617.2 + AY23, and AY24. Most clusters had the same strain within each patient, except cluster F. For cluster F, patient 16 was detected positive after regular screening because he took care of patient 15, which explains the distance between the dates of the SARS-CoV-2 positive results between the patients.

The above data might be easier to understand if presented in a table or added in the table 1. The annotation of the patient “patients six, seven, 11, and 20” should be written as Patient 6, 7, 11, and 20, as it is not counting.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

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Virology, molecular biology, and bioinformatics

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19 Jul 2024 | for Version 2

Pakorn Aiewsakun, Mahidol University, Bangkok, Thailand

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Not Approved

Summary of the article
This work by Ibrahim et al. (first published: 08/2022, latest published: 10/2023) presents an analysis of old SARS-CoV-2 genomes and clinical data collected from 22 patients across 8 families in Jakarta, Indonesia, between 01/2021-08/2021. In accordance with the journal's guidelines (https://f1000research.com/for-referees/guidelines), I will not evaluate its novelty or interest levels, but focus solely on the scientific validity of the article.

Comments:
The phylogenetic tree presented in the work is an UPGMA tree constructed using the Tamura-Nei substitution model. From a technical perspective, a maximum likelihood tree or Bayesian tree would be much more superior, and they are not at all difficult to construct (e.g. by using IQ-TREE or BEAST). The nucleotide substitution model should also be properly selected for the analysis (e.g. by using ModelFinder in IQ-TREE), but it is unclear from the text if the Tamura-Nei model is the best-fit model or not. In any case, the authors should reconstruct their tree using best practices for tree reconstruction.

With sequence data from the 22 patients alone, seeing samples from the same household clustering together on the tree does not immediately imply household transmission. To maximally rule out the possibility of (hidden) community-acquired infections, the authors should include as many community samples from the same time and location as possible in the phylogenetic analysis, and see if samples from the same household still cluster together.

In addition, given that the authors have sample collection dates, they should attempt to time-calibrate the tree to see if the estimated timescales are reasonable under the household transmission assumption or not. This additional temporal information would help further justify their conclusions.

Without more rigorous analysis, I feel that the authors should avoid concluding that what they saw were household transmissions despite suggestive patient histories. For example, in cluster F, although the behavioural history strongly suggests that patient 16 likely contracted the infection from patient 15, the phylogenetic tree indicates that they had completely different sources of infection. This gives a clear example of an unexpected result. Thus, the current household infection rates are scientifically unjustified. The authors should recompute and discuss these rates more carefully.

Regarding the statement:
“In clusters C (4 of 4), D(5 of 5), E (5 of 5), F (3 of 3),G(4 of 4), andH (4 of 4), the whole family had become infected with SARS-CoV-2. For cluster A, only three of eight family members became infected with COVID-19. For cluster B, 4 of 5 family members caught COVID-19.”
These numbers do not match those in Table 1 and sum to 32, not 22. Please clarify these results to avoid misunderstandings.

The results regarding cluster F should be discussed more. The current discussion stating “[f]or cluster F, patient 16 was detected positive after regular screening because he took care of patient 15, which explains the distance between the dates of the SARS-CoV-2 positive results between the patients” appears incomplete. It is not just the dates that differ but also the strains of the viruses. Please discuss more thoroughly.

Regarding the statement:
“Our study showed a ti/tv ratio decline between the GH and GK clades.”
Please elaborate on why the ti/tv ratio is particularly interesting to consider. Also, could this decline simply be due to the relatively small number of GK samples and their short collection period?

Regarding the statement:
“Our observation showed the SARS-CoV-2 transmission in the household setting through the workplace”
No, this conclusion is not supported by the results presented. To assert this, the authors need to also analyse samples from the patients’ workplaces, which are lacking from the current study. The results should be discussed more rigorously.

Based on the described methods, in addition to demographic and clinical data, the authors likely have RT-PCR CT values as well (?). Why were these data not analysed?

Table 2 description needs improvement. Also, either medians or IQRs are not reported in the table. Please address these issues.

Lastly, throughout the manuscript, there are many grammatical errors and awkwardly phrased sentences that, while grammatically correct, could be better written. Here are some specific examples from the introduction:
“It followed by the emergence of the variants of concern,..” could be revised to “This variant was then followed by the emergence of several other variants of concern,..”
“…sharing spaces with their healthy family member…” should be revised to “…sharing spaces with their healthy family members…”
“…encourage their employee to work from office…” should be revised to “…encourage their employees to work from office…”
Additionally, some statements are no longer relevant due to changes in current circumstances. Again, here are some specific examples:
“Lockdown and quarantine have been the main actions applied to contain the spread of SARS-CoV-2 in the population.”
“Although [using] masks [is] still mandatory in Indonesia,…”
“Therefore, preventive measures such as wearing a mask are still necessary, even after full vaccination”
Based on news updates I found on the internet, both lockdown and mask mandates were lifted in Indonesia since late December 2022, for example, making these sentences outdated.
The authors should carefully review their entire manuscript to correct all errors and revise the text and their discussion to better reflect the current situation.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Genomics, evolutionary analysis

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23 Aug 2022 | for Version 1

Serafeim C. Chaintoutis, Diagnostic Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece

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Not Approved

"Although lockdown had been applied to contain the disease, the household remains a critical place for its transmission." The two parts of the sentence are not combinable in a scientific manner. Several measures were applied to contain the disease. And many places remain critical for transmission. This also applies to the introduction.
"Naso-oropharyngeal swab..." Do authors mean both nasopharyngeal and oropharyngeal? Please elaborate.
"The infection rate ranged from 37.5 to 100%". You mean within families?
"Four breakthrough cases were detected..." Do authors mean vaccine breakthrough?

Introduction:

"D614G was the first reported mutation..." This is technically an amino-acid substitution. Please, be precise.
"This mutation then increased and generated the emergence of the variants of concern". How is this supported? This is scientifically unjustified.
"This study was performed to assess the variation of SARS-CoV-2 strains and their clinical manifestations within family clusters in Jakarta, Indonesia". By this, the impression that of large-scale study is being given. However, 8 families were only investigated. This is not representative. How were these family clusters chosen? Was there any reason? There might be a bias in the selection of families/research material, with potential false community representation results.

Materials and Methods:

Details on the sequencing methodology and analysis are missing. Herein, sequencing was performed using "Oxford Nanopore’s GridION sequencer", with read depth lower than Illumina or Ion Torrent systems usings critical to verify that the identified mutations are not an "artifact" of the sequencing process.

References:

Need to be checked and verified.

Fig. 1:

The relevant caption is missing several important pieces of information. What type of tree is this? Many trees are characterized as "phylogenetic trees" Details are needed on how this analysis was performed and the tree was constructed. This information needs to be added to the figure caption. The information provided on the branches is not easy to read and understand.

Tables:

Table 1. This table presents Pango lineages (i.e., B.1) that were not anticipated as of January 2021.
Table 2. It is not clear whether the authors mean in fact mutations, or substitutions by "total N mutations", "total S mutations", etc.
Table 3. This table also presents amino-acid substitutions, but they are named "mutations". Authors need to be precise.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Clinical microbiology, infectious diseases, immunology, virology

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Author Response

16 Oct 2023

Fera Ibrahim, Clinical Microbiology Department, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Dear Serafeim C. Chaintoutis,

Thank you very much for your time and effort to review our study.

Here's our response for each input:

Abstract:

"Although lockdown had been applied to contain the disease, the household remains a critical place for its transmission." The two parts of the sentence are not combinable in a scientific manner. Several measures were applied to contain the disease. And many places remain critical for transmission. This also applies to the introduction.

Response: Revised sentence: The lockdown had been applied to prevent transmission of virus, even the transmission in household remains a critical problem especially in poor and developing countries with more burden family.
"Naso-oropharyngeal swab..." Do authors mean both nasopharyngeal and oropharyngeal? Please elaborate.

Response: yes, it refer to nasopharyngeal and oropharyngeal.
"The infection rate ranged from 37.5 to 100%". You mean within families?

Response: yes, this rate resembles the infection rate in the families. Revised sentence: Therefore, the infection rate in family ranged from 37.5% to 100%.
"Four breakthrough cases were detected..." Do authors mean vaccine breakthrough?

Response: it means COVID19 occur even after the individual received a vaccine. Revised sentence: Four vaccine breakthrough cases were detected in this study, which were infected by the virus from GH and GK clade.

Introduction:

"D614G was the first reported mutation..." This is technically an amino-acid substitution. Please, be precise.

Response: it is an amino-acid substitution, thank you very much for the corrective comment; we will change this “mutation” in to “amino acid substitution” on whole manuscript.
"This mutation then increased and generated the emergence of the variants of concern". How is this supported? This is scientifically unjustified.

Response: we agree that mutation not always drive to the increased or generated novel voc, thus this sentence will be change in to “The mutation may increase and generate the emergence of the variants of concern”
"This study was performed to assess the variation of SARS-CoV-2 strains and their clinical manifestations within family clusters in Jakarta, Indonesia". By this, the impression that of large-scale study is being given. However, 8 families were only investigated. This is not representative. How were these family clusters chosen? Was there any reason? There might be a bias in the selection of families/research material, with potential false community representation results.

Response: samples on this study are from government program of COVID-19 tracking which RT-PCR diagnosis only taken place when symptoms are appears, thus it was limitations in this study.

Materials and Methods:

Details on the sequencing methodology and analysis are missing. Herein, sequencing was performed using "Oxford Nanopore’s GridION sequencer", with read depth lower than Illumina or Ion Torrent systems usings critical to verify that the identified mutations are not an "artifact" of the sequencing process.[P1]

Response: The methods for nanopore sequencing were done exactly as the protocols described in the citations (Citations number 10 and 11) and according to manufacturers instructions. For the depth, as manufacturers suggestions and based on the citations, the sequencing was set to achieve 100x depth of coverage across all targets. Therefore, all the sequencing results only can be proceeded for further analysis if the 100x depth of coverage was achieved. We determined the sequence was good enough for further analysis because we used more depth than required (50x) and run only 10 specimens per nanopore’s flowcells to achieve our desired coverage. We will add this information to the manuscript for the confirmation. In addition, sequencing using illumina platform was not feasible in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives.

Fig. 1:

The relevant caption is missing several important pieces of information. What type of tree is this? Many trees are characterized as "phylogenetic trees" Details are needed on how this analysis was performed and the tree was constructed. This information needs to be added to the figure caption. The information provided on the branches is not easy to read and understand.[P2]

Response: The phylogenetic tree was built using Geneious Prime 2021.1.1, using UPGMA as the tree build method and Tamura-Nei as the genetic distance model, with NC_045512 as the reference sequence. We had mentioned it into our manuscript.

Tables:

Table 1. This table presents Pango lineages (i.e., B.1) that were not anticipated as of January 2021.
Table 2. It is not clear whether the authors mean in fact mutations, or substitutions by "total N mutations", "total S mutations", etc.

Response: They refer to amino acid substitution within N or S protein.
Table 3. This table also presents amino-acid substitutions, but they are named "mutations". Authors need to be precise.

Response: Thank you very much for the corrective comments, the data presented in Table 3 are refer to amino acid substitutions, thus we will revise its “mutation” in to “amino acid substitution”.

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No competing interests were disclosed.

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29 Views

15 Aug 2022 | for Version 1

So Nakagawa, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan

29 Views Cite this report Responses(1)

Approved With Reservations

Introduction: “D614G” should be clearly explained when it first appeared.
Table 1: Are PANGO IDs isolated for Patients 4 to 6 “B.1”? The B.1 lineage is not common in January 2021 in the world. Please verify it.
Figure 1: The legend of the figure is missing, and I could not understand the meaning of the values shown for each branch. In addition, clades and PANGO IDs should be shown, which could be informative for readers.
Table 3: “Total S mutation” and “Total N mutation” are ambiguous. Do these include synonymous mutations as well?
References: Citations should be checked. For example, Reference #1 is not suitable for mentioning SARS-CoV-2 VOCs.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Genomics.

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Author Response

16 Oct 2023

Fera Ibrahim, Clinical Microbiology Department, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Dear So Nakagawa,

Thank you very much for your input and effort to review our paper.

Major comments

The details of each nanopore sequencing should be shown in the manuscript. For nanopore sequencing, the quality is known to be relatively low compared to Illumina sequencing. Therefore, if the depth is insufficient, it is impossible to distinguish whether those are mutations or sequencing errors. Please provide more information on the sequencing status (including depth, coverage, and any “shallow” regions if they exist) for each sample.[P1]

Response: Thank you very much for the inputs. We already use the cutoff 100x depth of coverage for all regions before we proceed for analysis. That’s also why we only included this small number of specimens, since only these specimens could pass such cutoff. In addition, sequencing using illumina platform was not feasible and too expensive in Indonesia for small project like ours during the period of our study, therefore we relied on Nanopore technology to support our country’s surveillance initiatives. Your message on Illumina is well noted, we will try them if we got more fundings in the future.

Minor comments

Introduction: “D614G” should be clearly explained when it first appeared.

Response: thank you for the input, it’s been revised.
Table 1: Are PANGO IDs isolated for Patients 4 to 6 “B.1”? The B.1 lineage is not common in January 2021 in the world. Please verify it.

Response : According to pango lineage page (Cov-Lineages, B.1 earliest date of detection was January 1^st,2020. Therefore, we’re sure that the B.1 lineage had arrived to Indonesia in 2021. We will add O’Toole’s study as part our methods in terms of evaluating the pangolins lineage.
Figure 1: The legend of the figure is missing, and I could not understand the meaning of the values shown for each branch. In addition, clades and PANGO IDs should be shown, which could be informative for readers.

Response : Thankyou for the input, the figure is revised.
Table 3: “Total S mutation” and “Total N mutation” are ambiguous. Do these include synonymous mutations as well?

Response : the mutations included only the non-synonymous one, as resulted from the GISAID analysis reports.
References: Citations should be checked. For example, Reference #1 is not suitable for mentioning SARS-CoV-2 VOCs.

Response: Thank you for the concerns. We consider your input and we had replaced them with different citations.

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Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Observation of SARS-CoV-2 genome characteristics and clinical manifestations within eight family clusters from GH and GK clades in Jakarta, Indonesia

Abstract

Keywords

Introduction

Methods

Ethics and patient sampling

Specimen collection and processing

Variant analysis and phylogenetic tree construction

Results

Table 1. Characteristics of the patients included in the study.

Figure 1. Phylogenetic tree of all the clusters.

Table 2. Bioinformatics analysis results between clade GH and GK.

Table 3. The distribution of the nonsynonymous mutations of the SARS-CoV-2 spike gene.

Discussion

Data availability

Acknowledgements

References

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