Strokach Aleksandra, Zoruk Polina, Boldyreva Daria, Morozov Maxim, Olekhnovich Evgenii, Veselovsky Vladimir, Babenko Vladislav, Selezneva Oksana, Zakharevich Natalia, Larin Andrey, Koldman Severina, Koldman Vail, Odorskaya Maya, Yunes Roman, Pavlov Vladislav, Kudryavtseva Anna, Danilenko Valeriy, Klimina Ksenia
Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia.
Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency, Moscow, Russia.
Front Microbiol. 2025 May 20;16:1584359. doi: 10.3389/fmicb.2025.1584359. eCollection 2025.
Advancements in sequencing technologies, such as Illumina and Oxford Nanopore Technologies (ONT), have significantly improved microbiome research. However, variations in sequencing platforms, primer selection, and DNA quality may influence microbial diversity assessments, particularly in studies of gut microbiota. This study systematically evaluates these factors in mouse gut microbiota analysis, comparing 16S rRNA gene sequencing and metagenome sequencing (MS) across both platforms.
Our findings highlight the critical influence of primer selection on 16S rRNA sequencing results, with certain primer combinations detecting unique taxa that others miss. Despite these variations in taxonomic resolution, all tested primer sets consistently revealed significant differences between experimental groups, indicating that key microbial shifts induced by bacterial cultures remain detectable regardless of primer choice. A comparative analysis of Illumina and ONT 16S rRNA sequencing revealed notable differences in microbial diversity profiling, with ONT capturing a broader range of taxa. In contrast, MS on both platforms showed a high degree of correlation, indicating that ONT sequencing errors have minimal impact on taxonomic diversity estimations. Furthermore, the type of extracted DNA (high molecular weight vs. standard DNA) had little on microbial diversity outcomes, underscoring the robustness of these sequencing technologies.
These results highlight the advantages and limitations of different sequencing strategies in microbiota research. While 16S rRNA sequencing remains a cost-effective tool for assessing bacterial diversity, MS provides superior taxonomic resolution and more precise species identification. Our study advocates for a hybrid approach that combines multiple sequencing technologies to achieve a more comprehensive and accurate representation of microbial communities.
测序技术的进步，如Illumina和牛津纳米孔技术（ONT），显著改善了微生物组研究。然而，测序平台、引物选择和DNA质量的差异可能会影响微生物多样性评估，尤其是在肠道微生物群研究中。本研究系统评估了小鼠肠道微生物群分析中的这些因素，比较了两种平台上的16S rRNA基因测序和宏基因组测序（MS）。
我们的研究结果突出了引物选择对16S rRNA测序结果的关键影响，某些引物组合能检测到其他组合遗漏的独特分类群。尽管在分类分辨率上存在这些差异，但所有测试的引物组都一致显示实验组之间存在显著差异，这表明无论引物选择如何，细菌培养诱导的关键微生物变化仍然可以检测到。对Illumina和ONT 16S rRNA测序的比较分析显示，微生物多样性谱存在显著差异，ONT捕获的分类群范围更广。相比之下，两种平台上的MS显示出高度相关性，表明ONT测序错误对分类多样性估计的影响最小。此外，提取的DNA类型（高分子量DNA与标准DNA）对微生物多样性结果影响不大，这突出了这些测序技术的稳健性。
这些结果突出了微生物群研究中不同测序策略的优缺点。虽然16S rRNA测序仍然是评估细菌多样性的一种经济有效的工具，但MS提供了更高的分类分辨率和更精确的物种鉴定。我们的研究提倡采用一种结合多种测序技术的混合方法，以更全面、准确地呈现微生物群落。
