Hong Bo-Young, Driscoll Mark, Gratalo Dawn, Jarvie Thomas, Weinstock George M
Dr. Kiran C. Patel College of Allopathic Medicine, Nova Southeastern University, Fort Lauderdale, FL 33328, USA.
Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA.
Int J Mol Sci. 2024 Mar 4;25(5):2966. doi: 10.3390/ijms25052966.
Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent ('Rapid' protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel 'Rapid' method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the 'Rapid' protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel 'Rapid' DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel 'Rapid' DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing.
下一代测序技术通过实现微生物群落水平的序列分析，推动了人类微生物组研究的快速发展。尽管所有微生物组测序方法都依赖于从样本中回收DNA作为第一步关键步骤，但裂解方法可能是微生物组图谱偏差的主要决定因素。基于温和酶的DNA制备方法可保留DNA质量，但可能因未能打开难以裂解的细菌而使结果产生偏差。像珠磨法这样的机械方法也可能使DNA回收产生偏差，因为打破更坚韧细胞壁所需的机械能可能会剪切更容易裂解的微生物的DNA，并且剪切程度可能因研磨时间和强度而异，从而影响可重复性。我们介绍了一种非机械、非酶促的新型快速微生物DNA提取程序，适用于基于16S rRNA基因的微生物组分析应用，该程序无需珠磨。将碱性、加热和去污剂同时应用于毫克量的样本（“快速”方案），在难裂解和易裂解细菌群体中提供了一致的代表性，等同于或优于现有方案，产生了足够的高质量DNA用于全长16S rRNA基因PCR。使用包含难裂解和易裂解细菌的模拟细菌群落对这种新型“快速”方法进行了评估。人类粪便样本测试将新型快速方法与标准人类微生物组计划（HMP）方案用于肺癌患者和对照的样本进行了比较。使用Illumina平台上的V1V3和V4区域以及PacBio平台上的V1V9区域的16S rRNA基因测序分析了从两种方法中回收的DNA。我们的研究结果表明，“快速”方案始终产生更高水平的厚壁菌门物种，更准确地反映了细菌群落结构的特征，这在模拟群落评估中得到了证实。这种新型“快速”DNA裂解方案减少了珠磨法和酶促裂解方法常见的群体偏差，为改进微生物群落分析提供了机会，同时将样本输入减少到10毫克或更少，并能够以标准平板形式快速转移和同时裂解96个样本。与广泛使用的商业方法相比，可以将样本处理时间减少2０倍，总体时间优势为2倍。我们得出结论，这种新型“快速”DNA提取方案为制备用于16S rRNA基因扩增子测序的粪便标本提供了一种可靠的替代方法。
