Comparison of DNA Extraction Methods in Analysis of Salivary Bacterial Communities

Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1–3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used.

非培养型高通量测序技术现已被广泛应用于细菌群落研究领域。尽管此类方法相较于传统培养法更具优势，但仍会在实验与生物信息学层面引入偏差。本研究通过两种不同方案提取宏基因组DNA（metagenomic DNA），并对其16S rDNA V1~3扩增子开展焦磷酸测序（pyrosequencing），以此评估人类唾液微生物组的多样性。两种提取方案分别为：仅使用蛋白酶K消化且未设置后续DNA净化步骤的方案，以及经珠磨式机械裂解后辅以柱式DNA纯化的方案。研究结果显示，两种提取方法的结果具有高度一致性，尤以微生物群落组成层面最为显著。在统一的生物信息学分析流程下，任一提取方案处理的样本中平均占比高于0.12%的所有分类单元（taxa），均可在另一提取方案处理的样本中被检测到。对于两种提取方案而言，相同的分类单元均呈现出丰度高、出现频率高的特征。隶属于放线菌门(Actinobacteria)、螺旋体门(Spirochaetes)、TM7、互养菌门(Synergistetes)以及柔膜菌门(Tenericutes)的序列读取(sequence reads)相对丰度，在机械裂解处理的样本中显著高于酶解法处理的样本；而厚壁菌门(Firmicutes)则呈现出相反的趋势。尽管机械裂解法的操作分类单元(operational taxonomic unit, OTU)丰富度更高，但两种提取方法在多样性指数方面并未呈现显著差异。提取流程本身的差异，相较于所采用的生物信息学分析流程带来的变异，影响更为显著。