Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127 Bonn, Germany.
Institute for Functional Gene Analytics, Bonn-Rhein-Sieg University of Applied Sciences, 53757 Sankt Augustin, Germany.
Int J Mol Sci. 2023 Jul 9;24(14):11262. doi: 10.3390/ijms241411262.
Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as and However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.
微生物组分析对于理解微生物的组成和多样性至关重要，但由于生物学和技术变量的影响，其解释往往具有挑战性。DNA 提取是一个关键步骤，它会极大地影响结果，尤其是在含有大量难以裂解的微生物的样本中。考虑到不同身体部位观察到的独特微环境，我们的研究旨在评估在不同临床样本类型中，DNA 提取过程中不理想的珠磨处理会在多大程度上引入偏差。问题是，对于某些样本类型，是否总是需要复杂的靶向提取方法来通过扩增子测序进行可靠的分类丰度估计，或者简单的替代方法是否有效。因此，对于四种不同的临床样本类型（粪便、宫颈拭子、皮肤拭子和医院表面拭子样本），我们比较了从针对每种样本类型的研究实验室中常规使用的靶向手动提取方案和专门为此目的设计的自动化提取方案中获得的结果。不出所料，我们发现对于粪便样本，需要使用剧烈的珠磨手动提取方案，以避免在所有研究的分类学水平上出现错误的分类比例，特别是重要属如 和 的错误低估或高估。然而，有趣的是，我们发现所有测试的方案在皮肤和宫颈拭子样本中都产生了相似的结果。我们的研究结果表明，实际自动化的程度在很大程度上取决于预期的微环境，皮肤和宫颈拭子比粪便样本更容易处理。在将本研究的结论扩展到分类丰度粗略估计以外的应用时，需要谨慎考虑。
