Metagenomic Analysis of the Oral Microbiome in Patients With Oral Lichen Planus: A Pilot Study
Conditions: Oral Lichen Planus
Sponsor: Kahramanmaras Sutcu Imam University

 
 
 
 
 
 
 
 
 
Date: 02.06.2023 
 
 
 
A PILOT STUDY ON THE MICROBIOLOGICAL DIVERSITY IN ORAL LICHEN PLANUS (OLP) 
Ayşe Gül ÖNER TALMAÇ, Gaye KESER, Hakan YÜLEK, Şükrü ÖNALAN, Filiz NAMDAR PEKİNER 
 
 
APPROACH AND METHODS TO BE APPLIED: 
 
Seven individuals who presented to the M.U. Faculty of Dentistry, Department of Oral and 
Maxillofacial Radiology Clinic and were diagnosed with "OLP" clinically and histopathologically 
(mucosa with OLP lesions and healthy mucosal tissue from the same patient) will constitute the 
patient group. Six healthy individuals who agreed to participate in the study and who presented 
to our clinic due to any complaints and who do not have any autoimmune symptoms will 
constitute the control group. 
 
Clinical Examination: 
1. Systemic and dental histories will be obtained for all patients included in the study. 
2. Oral mucosal and dental examinations will be performed for all patients included in the study. 
The location and type of lichen planus lesions, the type and condition of the dentures (if any), 
and the type and distribution of dental materials will be assessed and recorded on the "Patient 
History and Evaluation Form." 
 
Sample Collection 
 
1. Three samples will be collected from patients with OLP: Two from oral lichen planus lesions 
(OLP) located on the buccal mucosa and one from unaffected oral mucosa (vestibular sulcus or 
labial commissure) (H-OLP). 
2. Two samples will be collected from healthy patients: Two from bilateral healthy buccal 
mucosa (H). 
Participants will be advised to brush their teeth and avoid food intake for at least one hour before 
sampling. 
 
A 50 mg tissue sample will be collected from the patients using a cytobrash and transferred to 
2.0 ml centrifuge tubes. The tubes will be stored at -80°C for DNA extraction. 
 

DNA Isolation 
 
DNA extraction will be performed using the QIAmp DNA Mini Kit (Qiagen) using the QIACube Lt 
automated isolation device, according to the manufacturer's protocol. A 50 mg tissue scraping 
sample will be homogenized in 200 μL of 0.9% NaCl before adding proteinase K and lysis buffer, 
following the Purelink protocol. Isolated DNA will be measured at A260 absorbance using a 
Nanospectrophotometer (QIAxpert). If DNA amounts exceed 30 ng/μl, samples will be 
submitted for microbiota analysis. 
 
Microbiota analysis 
Targeted amplification from total genomic DNA will be performed using metabarcoding library 
construction using specific rDNA primers, equimolar pooling, MiSeq high-throughput 
sequencing (Illumina, San Diego, CA, USA), and multiplexing of paired-end reads (2 × 300 bp). 
Paired-end sequences will be clustered using the swarm clustering method, with a maximum of 
3 allowed differences between amplicons (d = 3). All clusters will be filtered for chimeras using 
VSEARCH. Operational Taxonomic Units (OTUs) with an abundance of less than 0.005% will be 
discarded because they are more likely to be chimeras from rare clusters. Finally, the affiliation 
of all bacterial OTUs will be verified using sequences from type material in the EzBioCloud 
database and the 16S rRNA and ITS databases for BlastN at NCBI. Remaining identified 
chimeras will be manually filtered, and these improved OTU tables will be used to calculate 
biostatistics using the PhyloSeq R package v1.28.0 implemented in FROGS. Alpha-diversity 
metrics (Chao1 and inverse Simpson indices) will be calculated after rarefaction. The 
abundance-based dissimilarity (beta-diversity) index, the Bray-Curtis index, will also be 
calculated after rarefaction. Microbiota analysis will be performed as a service outsourced. 
 
ANTICIPATED WORK DURATION: 1 year 
Start date: June 15, 2023 
Data evaluation period: 9 months 
End date: March 15, 2024 
 
 
Patient Consent Form 
 
Lichen Planus (LP) is a skin disease that can also occur inside the mouth. It can appear 
anywhere within the mouth, usually symmetrically on the inside of the cheeks, under and 
around the tongue, on the gums, and on the lips. It is called "Oral Lichen Planus" (OLP). It usually 
doesn't cause any symptoms, but sometimes a rough surface or burning sensation may be 
experienced. Although various factors are thought to be contributing factors, the exact cause is 
unknown. Therefore, various studies and studies are being conducted to diagnose and treat the 

disease. The study, titled "METAGENOMIC ANALYSIS OF ORAL LICHEN PLANUS AND RELATED 
ORAL PATHOGENS," will determine and evaluate the relationship between these factors and the 
disease. For this purpose, a 50 mg tissue sample will be collected from you using Cytobrash and 
transferred to 2.0 ml centrifuge tubes. 
 
In this study, samples will be collected from 12 individuals with oral lichen planus and 12 
healthy individuals and stored for analysis in a molecular analysis laboratory. This study aims to 
identify the relationship between oral lichen planus and oral pathogens, develop treatment 
options, and contribute to similar studies. 
 
Therefore, your CV, contact number, disease-related findings, and a swab sample will be 
collected from you. These will be used solely for this study, and the information obtained will be 
kept confidential. You are not responsible for any fees for this study. If you cease to be eligible 
for the study or voluntarily withdraw, there will be no interruption in your treatment at our clinic. 
 
I have been informed of these matters verbally or in writing. I have provided this form with the 
information provided. I have had the time and opportunity to ask relevant questions, and all my 
questions have been answered. I have read this form in its entirety. Under these conditions, I 
agree to participate in the clinical trial titled "METAGENOMIC ANALYSIS OF ORAL LICHEN 
PLANUS AND RELATED ORAL PATHOGENS" voluntarily, without any pressure or coercion. I also 
confirm the accuracy of all information I have provided about myself, including my medical 
history. 
 
Volunteer's 
Name-Surname Signature 
Address: Tel: 
Fax: 
Date: 
_____________________________________________________________________ 
Institutional official who witnessed the consent process from the beginning 
Name-Surname Signature 
Position: Tel: 
Fax: 
Date: 
 
Researcher making the statement 

Name-Surname: Signature 
Date: 
 
Dear Prof. Dr. Filiz NAMDAR PEKİNER informed me that a medical study would be conducted at 
the Department of Oral and Maxillofacial Radiology, Faculty of Dentistry, Marmara University, 
and provided me with the above information regarding this study. Following this information, I 
was invited to participate in this research as a "participant." 
 
I believe that if I participate in this research, the confidentiality of my information, which must 
remain between me and the doctor, will be treated with the utmost care and respect during this 
research. I have been given sufficient confidence that my personal information will be 
meticulously protected during the use of the research results for educational and scientific 
purposes. 
 
I may withdraw from the research without giving any reason during the project's implementation. 
However, I am aware that it would be appropriate to notify the researchers in advance of my 
withdrawal to avoid inconvenience. I also understand that I may be withdrawn from the research 
by the researcher to prevent any harm to my medical condition. I assume no financial 
responsibility for research expenses. I will not be reimbursed. I have been given the necessary 
assurance that any health problems I may experience, whether directly or indirectly, arising from 
the research will be treated with all necessary medical attention. I know that I will not be 
financially burdened with these medical interventions. 
 
I know that if I encounter a health problem during the research, I can contact Prof. Dr. Filiz 
NAMDAR PEKİNER at any time. I am not obligated to participate in this research and may choose 
not to participate. I have not been subjected to any coercive behavior to force me to participate. I 
also know that if I decline to participate, this will not compromise my medical care or my 
relationship with the physician. 
 
I will detail all information provided to me.