Plant Microbiosis Laboratory, Department of Microbiology, Friedrich Schiller University Jena, Jena, Germany.
Department of Microbial Interactions, IMIT/ZMBP, Eberhardt Karl University of Tübingen, Tübingen, Germany.
Mol Ecol Resour. 2021 Aug;21(6):1952-1965. doi: 10.1111/1755-0998.13408. Epub 2021 May 18.
Profiling diverse microbiomes is revolutionizing our understanding of biological mechanisms and ecologically relevant problems, including metaorganism (host + microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci has therefore become an instrumental tool for many researchers. Investigations in many systems are hindered, however, since essential sequencing depth can be lost by amplification of nontarget DNA from hosts or overabundant microorganisms. Here, we introduce "blocking oligos", a low-cost and flexible method using standard oligonucleotides to block amplification of diverse nontargets and software to aid their design. We apply them primarily in leaves, where exceptional challenges with host amplification prevail. A. thaliana-specific blocking oligos applied in eight different target loci reduce undesirable host amplification by up to 90%. To expand applicability, we designed universal 16S and 18S rRNA gene plant blocking oligos for targets that are conserved in diverse plant species and demonstrate that they efficiently block five plant species from five orders spanning monocots and dicots (Bromus erectus, Plantago lanceolata, Lotus corniculatus, Amaranth sp., Arabidopsis thaliana). These can increase alpha diversity discovery without biasing beta diversity patterns and do not compromise microbial load information inherent to plant-derived 16S rRNA gene amplicon sequencing data. Finally, we designed and tested blocking oligos to avoid amplification of 18S rRNA genes of a sporulating oomycete pathogen, demonstrating their effectiveness in applications well beyond plants. Using these tools, we generated a survey of the A. thaliana leaf microbiome based on eight loci targeting bacterial, fungal, oomycete and other eukaryotic microorganisms and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. This approach has potential to make questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems-based insights into microbial discovery. For fast and easy design for blocking oligos for any nontarget DNA in other study systems, we developed a publicly available R package.
对多样化微生物组的分析正在彻底改变我们对生物机制和生态相关问题的理解，包括元生物（宿主+微生物组）的组装、功能和适应。因此，对多个保守的、具有系统发育信息的基因座进行扩增子测序已成为许多研究人员的重要工具。然而，由于宿主或过度丰富的微生物中非目标 DNA 的扩增会导致必需的测序深度丢失，许多系统的研究受到阻碍。在这里，我们介绍了“阻断寡核苷酸”，这是一种使用标准寡核苷酸来阻断多种非目标物扩增的低成本、灵活的方法，以及一种辅助其设计的软件。我们主要将其应用于叶子中，那里普遍存在宿主扩增的特殊挑战。在八个不同的靶标基因座中应用拟南芥特异性阻断寡核苷酸可将不希望的宿主扩增减少多达 90%。为了扩大适用性，我们设计了通用的 16S 和 18S rRNA 基因植物阻断寡核苷酸，用于在不同植物物种中保守的靶标，并证明它们能够有效地阻断来自五个跨越单子叶植物和双子叶植物的五个目、五个物种的植物（Bromus erectus、Plantago lanceolata、Lotus corniculatus、Amaranth sp.、Arabidopsis thaliana）。它们可以在不影响 beta 多样性模式的情况下增加 alpha 多样性发现，并且不会损害植物衍生 16S rRNA 基因扩增子测序数据中固有的微生物负荷信息。最后，我们设计并测试了阻断寡核苷酸以避免扩增有孢子的卵菌病原体的 18S rRNA 基因，证明它们在植物以外的应用中非常有效。使用这些工具，我们基于靶向细菌、真菌、卵菌和其他真核微生物的八个基因座生成了拟南芥叶片微生物组的调查，并讨论了常用于描述叶片微生物组的扩增子测序区域的互补性。通过使扩增子测序更具针对性，这种方法有可能使各种研究系统中的问题更易于处理，从而更深入地了解微生物的发现，建立基于系统的见解。为了在其他研究系统中快速轻松地为任何非目标 DNA 设计阻断寡核苷酸，我们开发了一个公共可用的 R 包。
