Characterization of saliva microbiota’s functional feature based on metagenomic sequencing

Yang et al. SpringerPlus (2016) 5:2098 DOI 10.1186/s40064-016-3728-6 Open Access RESEARCH Characterization of saliva microbiota’s functional feature based on metagenomic sequencing Fang Yang1†, Kang Ning2†, Xiaowei Zeng2, Qian Zhou2, Xiaoquan Su2 and Xiao Yuan1* Abstract Objective: Saliva, a mixture of exocrinally secretive fluids, amounts to ~1.5 L daily and harbors numerous microbial inhabitants. However, except the organismal structure of saliva microbiota, the functional profile of saliva microbiota remain elusive. Methods: Here we used metagenomic sequencing to experimentally reconstruct the global genomic profile of saliva by sequencing total saliva DNA from two healthy and two caries-active (DMFT ≧ 6) adults. Results: We found that saliva microbiota, representing 30–60% of total saliva DNA in our samples, might carry functional signatures that were site-specific and caries-state-specific. Among microbiota from different hosts, a prominent functional core, but not an organismal core, was identified. Each microbiota exhibited functional redundancy where dominant genomes tend to encode more functional diversity yet without necessarily contributing to dominant functions. Furthermore, genetic polymorphisms of hosts were also unraveled from salivary DNA without apparent physical or sequence bias in human chromosomes. Conclusions: The microbial functional sensitivity to disease, links to specific functions, and permission of simultaneous genotyping of hosts and microbiota suggested sequencing salivary DNA might be an advantageous venue in uncovering both human and microbial basis of oral infections. Keywords: Caries, Function, Metagenome, Saliva, Microbiota, Whole-ecosystem sequencing Background Human body is the home to numerous symbiotic microbial organisms that with equal number of our own cells (Sender et al. 2016; Sleator 2010). They not just count upon the human body for food and shelter, but also provide the host with metabolic functions far beyond our own physiological capabilities (Sleator 2010). Thus, a “superorganism” view of human body suggested that interpretation on both human and their inhabitant microbiome holds potential for understanding the etiology of human diseases (Baquero and Nombela 2012). *Correspondence: † Fang Yang and Kang Ning contributed equally to this work 1 Department of Stomatology, Qingdao Municipal Hospital, Qingdao 266011, Shandong, China Full list of author information is available at the end of the article Saliva harbors a complex microbial community (Yang et al. 2012; Zarco et al. 2012). Multiple studies have characterized the organismal structure of saliva microbiota and revealing a high phylogenetic diversity (Keijser et al. 2008; Yang et al. 2012). Moreover, growing evidence from our research groups and others suggested that organismal structure of saliva microbiota is correlated with disease state such as caries (Yang et al. 2012), gingivitis (Huang et al. 2011) and periodontitis (Griffen et al. 2011), thus the organismal structure of saliva microbiota can potentially serve as a proxy to the oral health of the host (Nasidze et al. 2009; Kanasi et al. 2010). However, except the organismal structure of saliva microbiota, the functional profile of saliva microbiota remain elusive and it potential link to health and disease is not well understood (Human Microbiome Project Consortium 2012). Here we applied metagenomic sequencing on healthy (“H”) and caries-active (DMFT ≧ 6) (“C”) adults, aiming © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Yang et al. SpringerPlus (2016) 5:2098 to experimentally reconstruct the global genomic profile of saliva. The objective of this study was firstly to identify the percentage of microbial DNA contained in the saliva DNA samples and to investigate the functional signatures that related with host caries-status. Finally, genetic polymorphisms of hosts were also evaluated from those saliva samples. Although only four samples were generated for such a complex community, with our novel approach in combining sequence technology, combined assembly and various data analysis methods, our study may provide initial snapshot on how to sequencing the total saliva DNA to unravel both human and microbial basis of oral infections. Methods Study design All volunteers were given written informed consent in accordance with the sampling protocol with approval of the ethical committee of the Stomatology Hospital, Sun Yat-sen University. They were all unrelated individuals of both genders, aged between 18 and 23 years and shared a relatively homogeneous college-campus living environment in Sun Yat-sen University. To decipher the functional landscape of saliva, four saliva samples (two from the “healthy” individuals as H105, H114 and two from the “caries-active (DMFT ≧ 6)” subjects as C201, C218) were randomly selected for shotgun sequencing of total genomic DNA (Additional file 1: Table S1). Two boys of caries-active (DMFT ≧ 6) patients of DMFT equals to 7 and 9 were taken into this study. Moreover, two healthy individuals of one boy and one girl with DMFT of 0 were included in this study as healthy controls.“Healthy” individuals (DMFT = 0) and “caries-active (DMFT ≧ 6)” subjects (DMFT ≧ 6) were defined based on DMFT index, which measures the number of decayed, missing and filled teeth in epidemiologic surveys of dental caries (Anaise 1984). All reported no antibiotics intake for the preceding at least 6 months and no smoking or tobacco used. All were asked to avoid eating or drinking for 1 h before oral sampling. Those with other oral (for example, periodontitis or halitosis) or systematic diseases were excluded. Subjects were comfortably seated and, after a few minutes of relaxation, they were trained to avoid swallowing saliva and asked to lean forward and spit all the saliva they produced for 5 min into a graduated test tube. Participants were asked to collect at least 5 mL unstimulated saliva in a plastic cup and sample were transferred to a plastic tube and stored at −80 °C. Additional details of “Methods” section can be found in Additional file 2 Whole‑ecosystem sequencing of saliva For four of the saliva samples (two from the healthy hosts and the other two from caries-active (DMFT ≧ 6) hosts), shotgun pair-end libraries of total saliva genomic DNA Page 2 of 10 were prepared. Each metagenomic DNA libraries was then sequenced on one lane of pair-end 100 or 75 bp flow-cell on Solexa GA-IIx (Illumina, USA). Reads produced were processed via our computational pipelines customized for human oral microbiome analysis (Xie et al. 2010). All (...truncated)