Research Article Evaluation of Buccal Cell Samples for Studies of Oral Microbiota Guoqin Yu 1 , Steve Phillips 2 , Mitchell H. Gail 3 , James J. Goedert 4 , Michael Humphrys 5 , Jacques Ravel 5 , Yanfang Ren 2 , and Neil E. Caporaso 1 Abstract Background: The human microbiota is postulated to affect cancer risk, but collecting microbiota specimens with prospective follow-up for diseases will take time. Buccal cell samples have been obtained from mouthwash for the study of human genomic DNA in many cohort studies. Here, we evaluate the feasibility of using buccal cell samples to examine associations of human microbiota and disease risk. Methods: We obtained buccal cells from mouthwash in 41 healthy participants using a protocol that is widely employed to obtain buccal cells for the study of human DNA. We compared oral microbiota from buccal cells with that from eight other oral sample types collected by following the protocols of the Human Microbiome Project. Microbiota profiles were determined by sequencing 16S rRNA gene V3–V4 region. Results: Compared with each of the eight other oral samples, the buccal cell samples had significantly more observed species (P < 0.002) and higher alpha diversity (Shannon index, P < 0.02). The microbial communities were more similar (smaller beta diversity) among buccal cells samples than in the other samples (P < 0.001 for 12 of 16 weighted and unweighted UniFrac distance comparisons). Buccal cell microbial profiles closely resembled saliva but were distinct from dental plaque and tongue dorsum. Conclusions: Stored buccal cell samples in prospective cohort studies are a promising resource to study associations of oral microbiota with disease. Impact: The feasibility of using existing buccal cell collections in large prospective cohorts allows investigations of the role of oral microbiota in chronic disease etiology in large population studies possible today. Cancer Epidemiol Biomarkers Prev; 26(2); 249–53. Ó2016 AACR. Introduction Recent studies have revealed associations between the features of human microbial communities (human microbiota) and various diseases, including autoimmune disorders, diabetes, obesity, colon cancer, and even psychiatric conditions (1–7). Large prospective, population-based studies involving thousands of participants are needed to confirm those results and systematically study the role of the human microbiota in health. Profiles of the microbiota can be generated with next-generation DNA sequencing methods, but very few large long-term population-based cohorts have collected biological specimens for the purpose of microbiota research. Buccal cells, which have been obtained from mouthwash in many cohorts for the study of human genomic DNA, might be employed to study the oral microbiota. The purpose of the current study was to evaluate whether buccal cell specimens, collected by the protocol used in a prospective cancer cohort (http://prevention.cancer.gov/ major-programs/prostate-lung-colorectal), could be used for oral microbiota research by comparing buccal cell specimens with eight other oral sample types from the same subjects. Materials and Methods Participants Forty-three subjects were recruited at Eastman Institute of Oral Health, University of Rochester (Rochester, NY). All the subjects signed informed consent and filled out questionnaires. Results are based on 41 subjects after quality control–determined exclusions. Individuals with antibiotic usage or professional dental cleaning within the last 3 months or diagnosed with severe periodontal disease or cancer were excluded. The study was approved by Institutional Review Boards of the NCI (Rockville, MD) and University of Rochester (Rochester, NY). Sample collection and processing We collected 8 oral samples, including 2 dental plaque samples (supra- and subgingival plaque), raw saliva, and swabs from 5 soft tissue sites (keratinized gingiva, hard palate, buccal mucosa, palatine tonsil, and tongue dorsum) by following the protocols of the Human Microbiome Project (HMP; ref. 8; http://hmpdacc. org/doc/HMP_MOP_Version12_0_072910.pdf; see Supplemen- tary Fig. S1 for detailed sampling locations). In addition, buccal cells from mouthwash were also collected following the protocol 1 Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Bethesda, Maryland. 2 Eastman Institute of Oral Health, University of Rochester, Rochester, New York. 3 Biostatistics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Bethesda, Maryland. 4 Infections and Immunoepidemiology Branch, Divi- sion of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Bethesda, Maryland. 5 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland. Note: Supplementary data for this article are available at Cancer Epidemiology, Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). Corresponding Authors: Guoqin Yu, Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, NIH/NCI, Rockville, MD 20852. Phone: 240- 276-7249; Fax: 240-276-7832; E-mail: guoqin.yu@nih.gov; and Neil E. Caporaso, Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Rockville, MD, 20852. Phone: 240-276-7228; Fax: 240-276-7832; E-mail: caporasn@mail.nih.gov doi: 10.1158/1055-9965.EPI-16-0538 Ó2016 American Association for Cancer Research. Cancer Epidemiology, Biomarkers & Prevention www.aacrjournals.org 249 on June 8, 2020. © 2017 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from Published OnlineFirst October 21, 2016; DOI: 10.1158/1055-9965.EPI-16-0538