Comparison Of Salivary Microbial Genome Extraction And Amplicon Sequencing Data Analysis Methods

The oral cavity contains multiple microenvironments,each forming a complex microbial community that communicates through saliva.These microbes are closely related to the host’s health.Salivary microbiome research can be used to study the composition and function of microbial communities in human saliva and explore their relationship with oral health and disease.Therefore,the extraction and analysis of the salivary microbiome are particularly important in current oral biomedical research.The continuous development of metagenomic sequencing technology has greatly expanded the understanding of the relationship between the oral microbiome and systemic diseases.In this study,the process of saliva genomic DNA extraction was optimized by establishing a known mock community to find a rapid,low-cost,high-efficiency,and low-toxicity method of extracting genomic DNA from saliva samples,minimizing biases in DNA extraction and pursuing more accurate results.The main contents are as follows:(1)Utilizing the characteristics of the Veillonella genus,which lacks glucokinase and fructokinase,in combination with the ability of variant Streptococci to produce short-chain organic acids,we effectively isolated Veillonella parvula through syntrophic culturing and molecular identification.We separated Lactobacillus plantarum using selective culture media and confirmed it through whole-genome sequencing analysis.The two selected bacteria were added to a mock community composed of five microorganisms preserved in the laboratory(Candida albicans,Candida glabrata,Saccharomyces cerevisiae,Streptococcus sobrinus,and Streptococcus mutans).Finally,by measuring the growth curve and counting the various microorganisms in the mock community,we successfully obtained standard mock community samples.(2)The effects of different enzyme systems and different guanidine hydrochloride concentrations on DNA extraction using the guanidine hydrochloride enzyme magnetic bead method were investigated.It was found that the extraction effect was better when the dual enzyme system(a mixture of lysozyme and proteinase)was added at the beginning of the extraction process,and the extraction effect was best when the guanidine hydrochloride concentration was 6.2 M in the salt solution.Three magnetic bead-based genomic DNA extraction methods(commercial kit method,SDS magnetic bead method,and guanidine hydrochloride enzyme magnetic bead method)were compared using the mock community as the extraction object.The extraction results were evaluated in terms of concentration and purity,genomic electrophoresis,and PCR electrophoresis.The guanidine hydrochloride enzyme magnetic bead method showed relatively better extraction effects for both bacterial and fungal genomes.The SDS magnetic bead method was not suitable for the extraction of fungal genomic DNA.Therefore,in this study,the guanidine hydrochloride enzyme magnetic bead method had advantages in terms of genomic extraction quality and purity.(3)To further validate the superiority of the extraction methods,we performed16 S and ITS amplicon sequencing on the extracted genomic samples.Due to the massive variation in fungal copy numbers,only bacterial amplicon data from the mock community was used to assess the choice of tools for analyzing saliva samples.We found significant differences in the composition of the mock community species obtained using three different analysis tools: Deblur-VSEARCH,DADA2,and QIIME2.The results from DADA2 were more precise,aligning closer to the bacterial copy number ratios.Subsequently,amplicon sequences of saliva samples were analyzed using DADA2,revealing that Streptococcus,Prevotella,Neisseria,Lactobacillus,and Bacillus,among others,were the dominant bacterial genera,while Saccharomyces,Candida,and Cryptococcus were the dominant fungal genera in saliva.Analysis of alpha diversity revealed that in saliva genomes extracted using the guanidine hydrochloride magnetic bead method,the extraction effect for bacteria was similar to that of the kit method and was more stable.This method also achieved the highest abundance for fungal genomes,while the kit method had very limited extraction effect on some oral fungi,which were difficult to detect in amplicon sequencing analysis.Beta diversity analysis using Principal Coordinates Analysis(PCo A)showed no significant differences in bacterial genomic composition diversity between the guanidine hydrochloride magnetic bead method and the other two methods.However,there were significant differences in fungal genomic composition diversity between the kit method and the guanidine hydrochloride magnetic bead method.In conclusion,the guanidine hydrochloride enzyme magnetic bead method has advantages in extracting genomic DNA from saliva samples.It has a more significant effect on the extraction of the salivary microbiome,more comprehensively and accurately characterizing the composition of microbes in saliva.This provides a more effective method for the basic research of the oral microbiome.