Menzies Health Institute Queensland, Griffith University, Parklands Drive, Southport, QLD, 4215, Australia.
School of Pharmacy and Medical Sciences, Griffith University, Parklands Drive, Southport, QLD, 4215, Australia.
BMC Res Notes. 2023 Aug 11;16(1):169. doi: 10.1186/s13104-023-06451-7.
Growth in large population-based studies assessing contributions of the gut microbiota to health and disease requires high-throughput sample processing and analysis methods. This study assessed the impact that modifications to a commercially available magnetic bead based, semi-automated DNA extraction kit had on determination of microbial composition, relative to an established in-house method involving a combination of mechanical and chemical lysis. DNA was extracted from faecal samples from healthy adults (n = 12; 34-69 years), microbial composition was determined by V3-V4 16s rRNA sequencing and compared between extraction methods.
Diversity metrics did not differ between extraction methods. Differences in the relative abundance of key phyla, including a significantly lower abundance of the Firmicutes (p = 0.004) and higher relative abundance of the Bacteroidetes (p = 0.005) and Proteobacteria (p = 0.008) phyla were noted where the DNA extraction did not include additional chemical and mechanical lysis. Principal coordinate analysis of family and genera level data also suggested a potential for sample pre-processing to impact microbial composition. Observations of the potential for skewed microbial composition profiles from samples prepared using a semi-automated DNA extraction kit without additional sample pre-processing highlights a need for consideration of standardisation of methodological approaches to increase the comparability of microbial compositional data.
在评估肠道微生物群对健康和疾病的贡献的大型基于人群的研究中，需要高通量的样本处理和分析方法。本研究评估了对市售基于磁珠的半自动化 DNA 提取试剂盒进行修改对微生物组成的确定的影响，与涉及机械和化学裂解组合的内部方法相比。从健康成年人（n=12；34-69 岁）的粪便样本中提取 DNA，通过 V3-V4 16s rRNA 测序确定微生物组成，并在提取方法之间进行比较。
提取方法之间的多样性指标没有差异。在关键门的相对丰度上存在差异，包括厚壁菌门（Firmicutes）的丰度显著降低（p=0.004），拟杆菌门（Bacteroidetes）和变形菌门（Proteobacteria）的相对丰度更高（p=0.005 和 p=0.008），其中 DNA 提取不包括额外的化学和机械裂解。在科和属水平数据的主坐标分析中，还观察到样品预处理可能会影响微生物组成。使用没有额外样品预处理的半自动化 DNA 提取试剂盒制备的样品中存在潜在的微生物组成谱偏斜的观察结果，突出了需要考虑标准化方法学方法，以提高微生物组成数据的可比性。
