Veselovsky Vladimir, Romanov Mikhail, Zoruk Polina, Larin Andrey, Babenko Vladislav, Morozov Maxim, Strokach Aleksandra, Zakharevich Natalia, Khamidova Sadokatkhon, Danilova Albina, Vatlin Aleksey, Pavshintsev Vsevolod, Chenguang Feng, Tsybizov Denis, Mitkin Nikita, Galanova Olesya, Zakharenko Alexander, Golokhvast Kirill, Klimina Ksenia
Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia.
Siberian Federal Research Center of Agrobiotechnology RAS, Krasnoobsk, Russia.
Front Microbiol. 2025 Aug 6;16:1633360. doi: 10.3389/fmicb.2025.1633360. eCollection 2025.
Soil microbiome profiling is crucial for understanding microbial diversity and its roles in ecosystem functioning and agricultural productivity. Recent advancements in high-throughput sequencing, particularly Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), offer long-read sequencing capabilities that enhance microbial community analysis. In this study, we performed a comparative evaluation of 16S rRNA gene sequencing using Illumina (V4 and V3-V4 regions), PacBio (full-length and trimmed V3-V4/V4 regions), and ONT (full-length) to assess bacterial diversity in soil microbiomes. We analyzed three distinct soil types and applied standardized bioinformatics pipelines tailored to each platform. To ensure comparability, sequencing depth was normalized across platforms (10,000, 20,000, 25,000, and 35,000 reads per sample). Our results demonstrated that ONT and PacBio provided comparable bacterial diversity assessments, with PacBio showing slightly higher efficiency in detecting low-abundance taxa. Despite differences in sequencing accuracy, ONT produced results that closely matched those of PacBio, suggesting that ONT's inherent sequencing errors do not significantly affect the interpretation of well-represented taxa. Our study demonstrated that, regardless of the sequencing technology used and the choice of the target region (full-length 16S rRNA gene or its regions), microbial community analysis ensures clear clustering of samples based on soil type. The only exception is the V4 region, where no soil-type clustering is observed ( = 0.79). These results provide a comprehensive evaluation of sequencing platform performance.
土壤微生物群落分析对于理解微生物多样性及其在生态系统功能和农业生产力中的作用至关重要。高通量测序技术的最新进展，特别是太平洋生物科学公司（PacBio）和牛津纳米孔技术公司（ONT），提供了长读长测序能力，增强了微生物群落分析。在本研究中，我们使用Illumina（V4和V3-V4区域）、PacBio（全长和修剪后的V3-V4/V4区域）和ONT（全长）对16S rRNA基因测序进行了比较评估，以评估土壤微生物群落中的细菌多样性。我们分析了三种不同的土壤类型，并应用了针对每个平台量身定制的标准化生物信息学流程。为确保可比性，对各平台的测序深度进行了标准化（每个样本10,000、20,000、25,并35,000条 reads）。我们的结果表明，ONT和PacBio提供了可比的细菌多样性评估，PacBio在检测低丰度分类群方面效率略高。尽管测序准确性存在差异，但ONT产生的结果与PacBio的结果非常匹配，这表明ONT固有的测序错误不会显著影响对代表性良好的分类群的解释。我们的研究表明，无论使用何种测序技术以及目标区域（全长16S rRNA基因或其区域）的选择如何，微生物群落分析都能确保根据土壤类型对样本进行清晰的聚类。唯一的例外是V4区域，在该区域未观察到土壤类型聚类（ = 0.79）。这些结果对测序平台性能进行了全面评估。
