Optimization of Sampling for Molecular Microbial Research on Ocular Surface Samples and Building a Framework for Comparing Current Sequencing Results Using Different Extraction Protocols.
Phase: NA
Conditions: Microbiota, Ocular Surface
Sponsor: Universitaire Ziekenhuizen KU Leuven

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
Page 1 of 19 
 
 
 
Protocol Full Title: 
 
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
Protocol Acronym/short title: 
 
EPSO  
 
 
Version and date of protocol: 
 
 v 7.0 dd 01-02-2021 
 
Funding: 
 
Name: LRD/ KU Leuven: EFW-FOPRD1-O2010 (Perdaens Eye Research Fonds) 
Address: Waaistraat 6, 3000 Leuven 
 
Name: UZ Leuven: Fonds Academisch onderzoek, S62672 
Address: Herestraat 69, 3000 Leuven 
 
Principal Investigator: 
 
Name: dr. H. Delbeke 
Address: UZ Leuven campus Gasthuisberg, Ophthalmology Department, Herestraat 49, 
3000 Leuven 
Telephone: +32 16/ 33 29 20 
Email: heleen.delbeke@uzleuven.be 
 
 
Study protocol EPSO_ S62672 v 7.0  dd01.02.2021 
 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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Signatures 
 
 
 
 
 
 
 
 
 
 
 
___________ 
 
 
 
 
 
 
Principal Investigator 
 
 
 
 
Date: 01.02.2021 
 
Name: Dr. H. Delbeke 
 
 
 
 
 
 
 
 
 
 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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Table of Contents 
Table of Contents ....................................................................................................................... 4 
 
 
1. 
Background, rationale and novelty ..................................................................................... 6 
2. 
Trial objectives and design ................................................................................................. 8 
2.1 
Preparation, organization and management of cross-sectional study ...................... 8 
3. 
Selection and withdrawal of subjects ................................................................................ 11 
3.1 Inclusion criteria ......................................................................................................... 11 
3.2 Exclusion criteria ........................................................................................................ 11 
3.3 Expected duration of trial ............................................................................................ 11 
4. 
Trial procedures ................................................................................................................ 12 
4.1 
Summary of visits/procedures ................................................................................ 12 
4.2 
Supervision and responsibilities ............................................................................ 13 
4.2.1 
Insurance ......................................................................................................... 13 
4.2.2 
Informed consent ............................................................................................ 13 
5. 
Assessment of efficacy ..................................................................................................... 14 
6. 
Assessment of safety ......................................................................................................... 14 
7. 
Statistics ............................................................................................................................ 14 
7.1 Sample size ................................................................................................................. 15 
7.2 Randomisation ............................................................................................................ 15 
7.3 Analysis ....................................................................................................................... 15 
8. 
Quality assurance .............................................................................................................. 16 
9. 
Direct access to source data and documents ................................................................. 16 
10. 
Ethics and regulatory approvals .................................................................................... 16 
11. 
Data Handling ............................................................................................................... 16 
12. 
Data Management ......................................................................................................... 17 
13. 
Publication Policy ......................................................................................................... 17 
14. 
Financial Aspects .......................................................................................................... 17 
15. 
References ..................................................................................................................... 17 
16. 
Addendum…………………………………………………………………………….19 
 
 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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Study Synopsis  
 
Title of clinical trial 
 
 
Optimization of sampling for molecular 
microbial research on ocular surface samples 
and building a framework for comparing 
current sequencing results using different 
extraction protocols. 
 
 
Protocol Short Title/Acronym 
 
 
EPSO 
 
 
Funding 
 
 
 
LRD/ KU Leuven: EFW-FOPRD1-O2010 
(Perdaens Eye Research Fonds) 
UZ Leuven: Fonds Academisch onderzoek 
S62672 
 
 
Principal Investigator 
 
 
Dr. H. Delbeke 
Sponsor 
 
University Hospitals Leuven 
Herestraat 49 
3000 Leuven 
 
Purpose of clinical trial 
 
 
 
Optimization of ocular sampling for microbial 
research and to provide a framework for the 
interpretation of research results for ocular surface 
microbiome studies based on differences in DNA 
extraction procedures of complex microbial 
communities.  
 
 
Trial Design 
 
 
Exploratory study using biological samples    
Summary of eligibility criteria 
 
 
• 
≥ 18 years old 
• 
Willing to undergo sampling of the 
conjunctiva  
• 
Fluent in written and verbal Dutch 
• 
Capable of giving informed consent 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
Page 5 of 19 
 
 
 
Version and date of final protocol 
 
 
 
v 7.0 dd 01.02.2021 
 
 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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1. Background, rationale and novelty 
 
The human microbiome is the collective genomic information of the community of commensal, 
symbiotic and pathogenic microorganisms that share our body space. They play a role in 
metabolism, homeostasis and maturation of the immune system (1) 
Since the publication of the Human Microbiome Project in 2008, a lot of research on the 
presence of a core microbiome on different body parts such as gut, skin and mouth has been 
executed. More and more evidence is emanating that a dysbiotic microbiome can induce a 
variety of autoimmune and inflammatory diseases. Especially the relationship between the 
intestinal microbiome and different inflammatory diseases has been widely investigated (2).  
Since culture-based methods only reflect a subset of the microorganisms present in complex 
human associated microbial communities, it is necessary to additionally use culture 
independent methods to further investigate the presence of a “core” microbiome per microbial 
habitat (3). High-throughput sequencing, such as amplicon-based studies or metagenomic 
studies are the current way to go. Targeted amplicon studies, such as 16S rRNA amplicon 
sequencing, focus on marker genes and use these markers to reveal the composition and 
diversity of the microbiota. Part of the 16S rRNA gene is amplified by PCR with universal 
primers that recognize highly conserved regions of the gene. This region is then further 
sequenced. Metagenomic studies, as shotgun whole genome sequencing (WGS) use, instead of 
universal primers, random primers to sequence overlapping regions of the complete genome (4, 
5) 
Several studies have described the presence of a core ocular surface microbiome, but the 
difference in methods used makes them quite difficult to compare.  
In terms of used swabs, some studies use sterile cotton swabs (6-11) others nylon flocked swabs 
(2). Also, the use of anesthetics is subject for discussion. Some researchers use anesthetics as 
this would improve the sampling on subjects (11, 12). However, since Shin et al. (9) published 
a significant higher alpha diversity when no anesthetic was used, most sampling was performed 
without anesthesia. The latter publication did however not provide any information on timing 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
Page 7 of 19 
 
 
between application and sampling. Indeed, the described effect could also be due to the diluting 
effect of the drop itself. In any case, when using a topical anesthetic drop, the firmness of the 
sampling can be considered influenced.  Possibly there is a vertical stratification with the 
presence of transient species in the more superficial layers and a more resident commensal 
microbiota in the deeper layers (7).  
To compare the effect of anesthetic drops, we decided to sample volunteers undergoing 
general anesthesia for any type of surgery. The right and left eye of one person will be 
randomized for a drop of artificial tears (Thealoz duo, Théa Pharma, Clerrmont – Ferrand, 
Cedex 2, France) or a drop of topical anesthesia (Minims Oxybuprocaine Hydrochloride 
0.4%, Bausch and Lomb, Aubenas, Frankrijk). One drop of  Thealoz duo has the same 
weight as one drop of  Minims Oxybuprocaine Hydrochloride 0.4%, (both 0,02 g), this 
makes the diluting effect comparable. The executer will be blinded. The rationale for general 
anesthesia is that the sampling pressure/depth will be similar with or without topical 
anesthesia since the executer won’t be influenced by the patients reaction. By comparing both 
eyes of one person, the immunological and genetic background will be the same. Moreover, 
Shalabi et al. showed a high level of concordance in microbiome composition in the same-
person eyes (13). The effect of environmental factors, such as the effect of repeatedly sleeping 
on one side, will be minimalized by randomization. To have the possibility to take the most 
obvious confounding factors in account, subjects will be asked to fill in a small questionnaire 
(see addendum A). 
 
In gut and oral microbiome research, it is currently acknowledged that the used DNA 
extraction method plays a crucial role in the recovery of microbial taxa (14-16). However, 
researchers focusing on less studied human microbial niches, like the ocular surface, seem not 
yet to be aware of the impact thereof.  The ideal extraction protocol should reflect the microbial 
richness and diversity present on the eye as adequate as possible since the DNA extraction 
method affects the observed species diversity downstream. We aim at comprehensible 
integration of the results from the scarce microbiota studies published in this field, by 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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comparing different DNA extraction procedures used in these studies. By sampling both eyes 
of 7 volunteers on 7 different points in time, 6 extraction protocols will be compared, of which 
one with and without human depletion. (fig.1). In an attempt to reduce possible confounding 
factors of repetitive sampling, sampling will not be performed on consecutive days and all 
samples from the 7 subjects from a certain day will be tested with a different extraction protocol 
in each subject (as outlined in the below overview).  
Figure 1 
Although the immunological and genetic background in one person are the same and Shalabi 
et al. showed a high level of concordance in microbiome composition in the same-person eyes 
(13) , the environmental factors may differ between eyes. First of all, patients are known 
unilateral eye rubbers, with sometimes clinical consequences such as keratoconus (17). 
Secondly, repeatedly sleeping on one side can have an effect on the ocular surface microbiome 
as well due to rubbing of the eyelid against the pillow and nocturnal eyelid eversion (18). To 
further clarify the effect of environmental factors on both eyes within one individual, the 7 
volunteers mentioned before will be asked to fill in a questionnaire (cfr. addendum A). 
The ambition of this study is to make our research done in this area more robust since it will 
determine our method used in each future project. 
2. Trial objectives and design 
We aim at clarifying 1) if the reported effects of the use of anaesthetic drops (9) are due to 
dilution of samples or not, 2) how different DNA extraction protocols distort our view on ocular 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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surface microbiota composition and 3) the degree of similarity between left and right eye in the 
same person.  
 
2.1 Preparation, organization and management of this exploratory study  
 
Part 1: Effect of  the use of anaesthetic drops on ocular microbial composition using 16S 
rRNA sequencing. 
 
Study cohort:  
Thirty volunteers undergoing general anaesthesia for any type of surgery will be informed 
about the study upon their consultation at the Ophthalmology Department of the University 
Hospitals Leuven, Belgium. In case a candidate is interested in participating, he/she will be 
informed about the study goals and protocols by dr. H. Delbeke or the responsible clinical 
research coordinator Inge Vriens during a personal conversation. Each volunteer will receive 
an information letter to take home.  
 
Study procedure:  
After signing the informed consent, each patient will be asked to fill in a questionnaire (cfr. 
addendum A). 
All participants will undergo conjunctival sampling of both eyes under general anaesthesia. 
The research coordinator or operating room nurse will applicate one drop of preservative free 
artificial tears to one eye Thealoz duo, Théa Pharma, Clerrmont – Ferrand, Cedex 2, 
France) and one drop of preservative free anaesthetics (Minims Oxybuprocaine 
Hydrochloride 0.4%, Bausch and Lomb, Aubenas, Frankrijk) to the other eye. A timed 5 
minutes will be in between application of the drop and the sampling. The sampling will be 
blinded for the executer and will be performed by myself, Heleen Delbeke, and/or residents 
and/ or medical students after proper training of correct sampling. Each eye will be sampled 
with a single, sterile, nylon, flocked swab (FLOQSwabs®; Copan, Brescia, Italy). The 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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sampling will be done from the nasal to temporal inferior conjunctival sac, the swab will be 
swirled in the opposite direction of the sampling itself. The swabs will be placed in a 
microcentrifuge tube and immediately frozen at −20 °C for a maximum period of 2 weeks, 
after which they will be transferred to a −80 °C freezer until further processing. DNeasy 
PowerSoil Kit will be used for DNA extraction. This extraction protocol is chosen based on 
its frequency used in ocular surface microbiome research. The extracted DNA will be 
analysed using the V4 region of the 16S rRNA gene. All steps in between sample taking and 
sequencing will be executed at the REGA institute for medical research or department of 
Biochemistry and Microbiology of the University of Gent. The sequencing itself will be done at a 
sequencing facility.  
 
Part 2: Clarification on how different DNA extraction protocols distort our view on ocular 
surface microbiota composition. 
 
Study cohort:  
Seven volunteers will be recruited using a poster at the Ophthalmology Department of the 
University Hospitals Leuven, Belgium and at the faculty of medicine, Catholic University 
Leuven, Belgium (cfr. addendum B). In case a candidate is interested in participating, he/she 
will be informed about the study goals and protocols by dr. H. Delbeke or the responsible 
clinical research coordinator Inge Vriens during a personal conversation. Each volunteer will 
receive an information letter to take home.  
 
Study procedure:  
After signing the informed consent, all eligible participants will be asked to fill in a small questionnaire (cfr. 
addendum A), after which they will undergo conjunctival sampling on 7 different points in time (figure 1). Based 
on the results of the first part of the study we will not use anaesthetic drops. 
There is no need for general anaesthesia. The sampling will be performed by myself, Heleen 
Delbeke, and/or residents and/ or medical students after proper training of correct sampling. 
Each eye will be sampled with a single, sterile, nylon, flocked swab (FLOQSwabs®; Copan, 
Brescia, Italy). The sampling will be done from the nasal to temporal inferior conjunctival sac, 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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the swab will be swirled in the opposite direction of the sampling itself. The swabs will be 
placed in a microcentrifuge tube and immediately frozen at −20 °C for a maximum of 2 weeks, 
after which they will be transferred to a −80 °C freezer until processing. Six extraction protocols 
will be compared, of which one with and without human depletion. The extracted DNA will be 
analysed using the V4 region of the 16S rRNA gene. All steps in between sample taking and 
sequencing will be executed at the REGA institute for medical research or department of 
Biochemistry and Microbiology of the University of Gent The sequencing itself will be done at a 
sequencing facility.  
 
Part 3: What is the degree of similarity between left and right eye in the same person.  
 
Study cohort and procedure:  
Part 3 is based on the questionnaire and sampling results of the seven subjects used in part 2. 
3. Selection and withdrawal of subjects 
 
3.1 Inclusion criteria 
 
Part 1: Effect of  the use of anaesthetic drops on ocular microbial composition using 16S 
rRNA sequencing. 
Subject has to be older than 18 years old 
 
Subject undergoes general anaesthesia for whatever reason   
The subject is willing to undergo sampling of the conjunctiva of both eyes 
The subject is fluent in written and verbal Dutch 
The subject is capable of giving informed consent 
 
Part 2: Clarification on how different DNA extraction protocols distort our view on ocular 
surface microbiota composition. 
 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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Subject has to be older than 18 years old 
 
The subject is willing to undergo sampling of the conjunctiva of both eyes on 7 different time 
points 
The subject is fluent in written and verbal Dutch 
The subject is capable of giving informed consent 
3.2 Exclusion criteria 
 
- Allergy to Oxybuprocainehydrochloride  
- Ocular surface disease 
- Medication usage in one eye only 
3.3 Expected duration of trial 
 
Inclusion of subjects is planned between August the 15th 2019 and the end of December 2020.  
4. Trial procedures 
4.1 Summary of visits 
 
Part 1: Effect of  the use of anaesthetic drops on ocular microbial composition using 16S 
rRNA sequencing. 
The conjunctival samples will take place at time of the planned general anesthesia. No 
additional visits are necessary.  
 
Part 2: Clarification on how different DNA extraction protocols distort our view on ocular 
surface microbiota composition. 
The conjunctival samples will take place at the Ophthalmology Department of the University 
Hospitals Leuven, Belgium on 7 different points in time. Sampling will take less than 5 
minutes. To compensate for travel expenses and time invested, each participant will receive at 

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Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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the end of their participation a voucher of 50 euros. 
 
4.2 Supervision and responsibilities 
 
The study will be performed under the supervision of dr. H. Delbeke. The subjects will be 
recruited at the Ophthalmology Department of the University Hospitals Leuven, Belgium and 
at the faculty of medicine, Catholic University Leuven, Belgium. The sampling will be 
performed by myself, Heleen Delbeke, and residents and/ or medical students. Each study 
subject will sign an informed consent. The participating investigators will perform their part 
of the study fully in accordance with the terms of the Protocol, the applicable national and 
international laws (amongst others: the European General Data Protection Regulation of  May 
25, 2018) and apply and adhere to regulations and rules as, amongst others, the Declaration of 
Helsinki (2008) and ICH GCP Guidelines. 
 
4.2.1 Insurance 
 
In accordance with the Belgian Law relating to experiments on human persons dated May 7, 
2004, UZ/KULeuven shall assume, even without fault, the responsibility of any damages 
incurred by a Study Patient  from the UZ/KU Leuven site and linked directly or indirectly to 
the participation to the Study, and shall provide compensation therefore through their 
insurance (Amlin Corporate Insurance, polisnr. 299.053.700, Vanbreda Risk & Benefits, 
Plantin en Moretuslei 297, 2140 Antwerp). 
 
4.2.2 Informed consent 
 
The Participating Site acknowledges that the Study can and will be conducted only on the basis 
of prior informed consent by the Subjects, or their legal representatives, to participate in the 
Study. The Participating Site shall obtain a signed informed consent form (ICF) for all patients 
prior to their enrollment and participation in the Study in compliance with all applicable laws, 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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regulations and the approval of the local Ethics Committee, if required. The Participating Site 
shall retain such ICFs in accordance with the requirements of all applicable regulatory agencies 
and laws. 
5. Assessment of efficacy 
Not applicable 
6. Assessment of safety 
 
When sampling is not performed under general anesthesia, a slight discomfort can be perceived. 
 
When  topical anaesthetics are being used, a transient stinging and blurring of the vision after 
instillation can occur. When used in larger amounts, epitheliopathy of the cornea can be noticed. 
This is only rare in the case of one drop. Systemic absorption will be reduced by asking the 
patient to gently close the lids and to press on the medial canthus for a minute following the 
instillation of the drops. There are no known interactions with other medical products. It will 
not be used during pregnancy or lactation, nor will it be used in patients with a known 
hypersensitivity to the product. 
 
After sampling the conjunctiva, a minor bleeding can occur in the conjunctival sac which can 
induce a feeling of bruising of the tissue. Scarring due to the intervention is extremely rare. 
7. Statistics 
7.1 Sample size 
 
Since this is an exploratory study, we are not able to perform proper sample sizing. 
For the first part, we aim at sampling 30 patients (60 samples) taking into account a maximal 
loss of 10 samples due to processing issues. Taking this loss into account, we will be able to 

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Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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process 50 samples to elucidate if the use of anesthetic drops has an effect on the sequencing 
results. 
For the second part, we wish to sample 7 volunteers so we can compare 7 extraction protocols 
on different subjects on non-consecutive days. Each protocol will be tested on a sample form 
a different individual on a different day (figure 1) to minimize possible confounding factors. 
In gut and oral microbiome research, samples are homogenized to make comparing of 
different extraction protocols possible. In ocular surface samples, the retrieved sample is to 
small for homogenization.  
 
7.2 Randomization 
 
A 30-patient randomization list will be generated by our clinical research coordinator  
using https://www.randomizer.org, where “1” will be a drop of preservative free anaesthetics 
in the right eye and “2” a drop of preservative free anaesthetics in the left eye.  
By using this software, we are able to automate the randomization, this to reduce bias due to 
manual randomization. When analysing our results, we will search for other biases due to 
randomisation, which we will be taken into account. 
 
 

Study protocol EPSO v 7.0 dd 01.02.2021:   
Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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7.3 Analysis 
 
The sequences will be analysed using the latest bioinformatics tools (like Dada2) and analysis 
will be done with the R software. The alpha diversity will be evaluated using Shannon’s 
Diversity index and Simpson’s diversity index; the beta diversity will be assessed by using 
Bray-Curtis dissimilarity, Jaccard distance and permutational multivariate analysis of variance. 
Differences will be considered statistically significant with a P-value ≤ 0.05. (19, 20) 
8. Quality assurance 
 
To assure maximal quality and reproducibility, the trial protocol will be followed rigorously. 
9. Direct access to source data and documents 
 
Only data gathered in the context of the trial will be used.  
10. 
Ethics and regulatory approvals 
 
The trial will be conducted in compliance with the principles of the Declaration of Helsinki 
(2008), the principles of Good Clinical Practice and in accordance with all applicable 
regulatory requirements. This protocol and related documents will be submitted for review 
to Ethics Committee of the University Hospitals Leuven. 
The Study will be conducted only on the basis of prior informed consent by the Subjects to 
participate in the Study. We shall obtain a signed informed consent form (ICF) for all 
patients prior to their enrollment and participation in the Study in compliance with all 
applicable laws, regulations and the approval of the (local) Ethics Committee, if required. 
We shall retain such ICFs in accordance with the requirements of all applicable regulatory 
agencies and laws. 
11. 
Data Handling 
 

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Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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We shall treat all information and data as confidential and shall not disclose such information 
to any third parties or use such information for any purpose other than the performance of the 
Study. The collection, processing and disclosure of personal data, will comply with applicable 
personal data protection and the processing of personal data (the European General Data 
Protection Regulation of May 25, 2018). We will protect the data from disclosure outside the 
research according to the terms of the research protocol and the informed consent document. 
The subject’s name or other identifiers will be stored separately from their research data and 
replaced with a unique code to create a new identity for the subject. 
12. 
Data Management 
 
All data will be analyzed and stored in a coded fashion, with a unique anonymous identifier for 
every subject. In the future, these data could be used in the context of this project, and for related 
projects. 
13. 
Publication Policy 
 
Any publication will be submitted to all co-authors at least thirty (30) days prior to submission 
or disclosure.  
14. 
Financial Aspects 
 
No commercial conflict of interest is related to this study. 
For the first part of this study, conjunctival sample will take place at time of the planned general 
anesthesia. No additional visits are necessary. Volunteers do not receive a financial 
compensation for their participation.  
For the second part, the subject needs to come on 7 different points in time for sampling. 
Sampling will take less than 5 minutes. The visit will not be charged. If there were to be 
additional medical expenses (medication and  visits) arising from participation in the study, 

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Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
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they will not be charged to the volunteer. To compensate for travel expenses and time invested, 
each participant will receive at the end of their participation a voucher of 50€.  
15. 
References 
 
1. 
Zaheer M, Wang CJ, Bian F, Yu ZY, Hernandez H, de Souza RG, et al. Protective role 
of commensal bacteria in Sjogren Syndrome. Journal of Autoimmunity. 2018;93:45-56. 
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Ozkan J, Nielsen S, Diez-Vives C, Coroneo M, Thomas T, Willcox M. Temporal 
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Singapore Health and Biomedical Congress, SHBC 2013. Annals of the Academy of 
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Schabereiter-Gurtner C, Maca S, Rolleke S, Nigl K, Lukas J, Hirschl A, et al. 16S 
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Dong Q, Brulc JM, Iovieno A, Bates B, Garoutte A, Miller D, et al. Diversity of bacteria 
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Ham B, Hwang HB, Jung SH, Chang S, Kang KD, Kwon MJ. Distribution and Diversity 
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Graham JE, Moore JE, Jiru X, Moore JE, Goodall EA, Dooley JS, et al. Ocular pathogen 
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Huang Y, Yang B, Li W, de Paiva CS, Jones DB, Stern ME, et al. Defining the normal 
core microbiome of conjunctival microbial communities 
Altered Mucosal Microbiome Diversity and Disease Severity in Sjogren Syndrome. Clin 
Microbiol Infect. 2016;22(7):643 e7- e12. 
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Shalabi NM, Tuzhikov AI, Galor A, Dong Q, Panchin AU, Thanathanee O, et al. 
Experimental approaches in the analysis of microbial community in ocular samples. 
Investigative Ophthalmology and Visual Science. 2014;55(13):1976. 

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Optimization of sampling for molecular microbial research on ocular surface samples and 
building a framework for comparing current sequencing results using different extraction 
protocols. 
 
 
 
 
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14. 
Costea PI, Zeller G, Sunagawa S, Pelletier E, Alberti A, Levenez F, et al. Towards 
standards for human fecal sample processing in metagenomic studies. Nat Biotechnol. 
2017;35(11):1069-76. 
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Wesolowska-Andersen A, Bahl MI, Carvalho V, Kristiansen K, Sicheritz-Ponten T, 
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 Addenda 
 
Addendum A: questionnaire to be filled in by all subjects