Metabarcoding of environmental DNA (eDNA) and DNA extracted from bulk specimen samples is a powerful tool in studies of biodiversity, diet and ecological interactions as its inherent labelling of amplicons allows sequencing of taxonomically informative genetic markers from many samples in parallel. However, the occurrence of so‐called ‘tag‐jumps’ can cause incorrect assignment of sequences to samples and artificially inflate diversity. Two steps during library preparation of pools of 5ʹ nucleotide‐tagged amplicons have been suggested to cause tag‐jumps: (a) T4 DNA polymerase blunt‐ending in the end‐repair step and (b) postligation PCR amplification of amplicon libraries. The discovery of tag‐jumps has led to recommendations to only carry out metabarcoding PCR amplifications with primers carrying twin‐tags to ensure that tag‐jumps cannot result in false assignments of sequences to samples. As this increases both cost and workload, a metabarcoding library preparation protocol which circumvents the two steps that causes tag‐jumps is needed. Here, we demonstrate Tagsteady, a PCR‐free metabarcoding Illumina library preparation protocol for pools of nucleotide‐tagged amplicons that enables efficient and cost‐effective generation of metabarcoding data with virtually no tag‐jumps. We use pools of twin‐tagged amplicons to investigate the effect of T4 DNA polymerase blunt‐ending and postligation PCR on the occurrence of tag‐jumps and demonstrate that both blunt‐ending and postligation PCR, alone or together, can result in detrimental amounts of tag‐jumps (here, up to ca. 49% of total sequences), while leaving both steps out (the Tagsteady protocol) results in amounts of sequences carrying new combinations of used tags (tag‐jumps) comparable to background contamination.

中文翻译：

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Tagsteady：一种元条形码文库制备协议，可避免错误地将序列分配给样本。

环境 DNA (eDNA) 和从大量样本样本中提取的 DNA 的元条形码是研究生物多样性、饮食和生态相互作用的有力工具，因为其固有的扩增子标记允许对来自许多样本的分类信息遗传标记进行并行测序。然而，所谓的“标签跳跃”的发生会导致序列对样本的错误分配并人为地增加多样性。已建议在 5ʹ 核苷酸标记的扩增子库的文库制备过程中的两个步骤会导致标签跳跃：（a）末端修复步骤中的 T4 DNA 聚合酶平端和（b）扩增子文库的连接后 PCR 扩增。标签跳转的发现导致建议仅使用带有双标签的引物进行元条形码 PCR 扩增，以确保标签跳转不会导致错误的序列分配到样本。由于这会增加成本和工作量，因此需要一种元条形码库准备协议来规避导致标签跳转的两个步骤。在这里，我们展示了 Tagsteady，这是一种无 PCR 的宏条形码 Illumina 文库制备协议，用于核苷酸标记的扩增子池，可以高效且经济地生成宏条形码数据，几乎没有标签跳转。我们使用双标记扩增子池来研究 T4 DNA 聚合酶平末端和连接后 PCR 对标记跳跃发生的影响，并证明平末端和连接后 PCR，单独或一起，