Schnell Ida Baerholm, Bohmann Kristine, Gilbert M Thomas P
Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, 1350, Copenhagen K, Denmark.
Center for Zoo and Wild Animal Health, Copenhagen Zoo, 2000, Frederiksberg, Denmark.
Mol Ecol Resour. 2015 Nov;15(6):1289-303. doi: 10.1111/1755-0998.12402. Epub 2015 Mar 20.
Metabarcoding of environmental samples on second-generation sequencing platforms has rapidly become a valuable tool for ecological studies. A fundamental assumption of this approach is the reliance on being able to track tagged amplicons back to the samples from which they originated. In this study, we address the problem of sequences in metabarcoding sequencing outputs with false combinations of used tags (tag jumps). Unless these sequences can be identified and excluded from downstream analyses, tag jumps creating sequences with false, but already used tag combinations, can cause incorrect assignment of sequences to samples and artificially inflate diversity. In this study, we document and investigate tag jumping in metabarcoding studies on Illumina sequencing platforms by amplifying mixed-template extracts obtained from bat droppings and leech gut contents with tagged generic arthropod and mammal primers, respectively. We found that an average of 2.6% and 2.1% of sequences had tag combinations, which could be explained by tag jumping in the leech and bat diet study, respectively. We suggest that tag jumping can happen during blunt-ending of pools of tagged amplicons during library build and as a consequence of chimera formation during bulk amplification of tagged amplicons during library index PCR. We argue that tag jumping and contamination between libraries represents a considerable challenge for Illumina-based metabarcoding studies, and suggest measures to avoid false assignment of tag jumping-derived sequences to samples.
在第二代测序平台上对环境样本进行代谢物条形码分析已迅速成为生态研究的一项重要工具。该方法的一个基本假设是能够将带标签的扩增子追溯到其来源的样本。在本研究中，我们解决了代谢物条形码测序输出中存在的标签错误组合（标签跳跃）导致的序列问题。除非能够识别这些序列并将其从下游分析中排除，否则标签跳跃会产生具有错误但已使用的标签组合的序列，从而导致序列错误地分配到样本中，并人为地夸大多样性。在本研究中，我们通过分别用带标签的通用节肢动物引物和哺乳动物引物扩增从蝙蝠粪便和水蛭肠道内容物中获得的混合模板提取物，记录并研究了Illumina测序平台上代谢物条形码研究中的标签跳跃现象。我们发现，在水蛭和蝙蝠饮食研究中，平均分别有2.6%和2.1%的序列具有可由标签跳跃解释的标签组合。我们认为，标签跳跃可能发生在文库构建过程中带标签扩增子池的平端化过程中，以及文库索引PCR过程中带标签扩增子大量扩增时形成嵌合体的结果。我们认为，标签跳跃和文库之间的污染对基于Illumina的代谢物条形码研究构成了相当大的挑战，并提出了避免将标签跳跃衍生的序列错误分配到样本中的措施。
