Toto Francesca, Scanu Matteo, Gramegna Maurizio, Putignani Lorenza, Del Chierico Federica
Unit of Microbiome, Bambino Gesù Children's Hospital, IRCCS, 00144 Rome, Italy.
Technogenetics S.p.A., 26900 Lodi, Italy.
Int J Mol Sci. 2025 May 29;26(11):5226. doi: 10.3390/ijms26115226.
High-throughput 16S rRNA metagenomic sequencing has advanced our understanding of the gut microbiome, but its reliability depends on upstream processes such as DNA extraction and bacterial library preparation. In this study, we evaluated the impact of three different DNA extraction methods (a manual method with an ad hoc-designed pre-extraction phase (PE-QIA), and two automated magnetic bead-based methods (T180H and TAT132H)) and two bacterial library preparation protocols ( and VeriFi) on the 16S rRNA-based metagenomic profiling of faecal samples. T180H and TAT132H produced significantly higher DNA concentrations than PE-QIA, whereas TAT132H yielded DNA of lower purity compared to the others. In the taxonomic analysis, PE-QIA provided a balanced recovery of Gram-positive and Gram-negative bacteria, TAT132H was enriched in Gram-positive taxa, and T180H was enriched in Gram-negative taxa. An analysis of Microbial Community Standard (MOCK) samples showed that PE-QIA and T180H were more accurate than TAT132H. Finally, the VeriFi method yielded higher amplicon concentrations and sequence counts than the protocol, despite the high level of chimeras. In conclusion, a robust performance in terms of DNA yield, purity, and taxonomic representation was obtained by PE-QIA and T180H. Furthermore, it was found that the impact of PCR-based steps on gut microbiota profiling can be minimized by an accurate bioinformatic pipeline.
高通量16S rRNA宏基因组测序增进了我们对肠道微生物群的了解，但其可靠性取决于DNA提取和细菌文库制备等上游流程。在本研究中，我们评估了三种不同的DNA提取方法（一种带有专门设计的提取前阶段的手动方法（PE-QIA），以及两种基于磁珠的自动化方法（T180H和TAT132H））和两种细菌文库制备方案（以及VeriFi）对粪便样本基于16S rRNA的宏基因组分析的影响。T180H和TAT132H产生的DNA浓度显著高于PE-QIA，而与其他方法相比，TAT132H产生的DNA纯度较低。在分类分析中，PE-QIA能平衡回收革兰氏阳性菌和革兰氏阴性菌，TAT132H富含革兰氏阳性分类群，而T180H富含革兰氏阴性分类群。对微生物群落标准（MOCK）样本的分析表明，PE-QIA和T180H比TAT132H更准确。最后，尽管嵌合体水平较高，但VeriFi方法产生的扩增子浓度和序列数量高于方案。总之，PE-QIA和T180H在DNA产量、纯度和分类代表性方面表现出色。此外，研究发现，通过准确的生物信息学流程可以将基于PCR的步骤对肠道微生物群分析的影响降至最低。
