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 5’ nucleotide-tagged amplicons have been suggested to cause tag-jumps; i) T4 DNA polymerase blunt-ending in the end-repair step and ii) post-ligation 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 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 post-ligation PCR on the occurrence of tag-jumps. We demonstrate that both blunt-ending and post-ligation 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.

Please see the paper and associated supporting information.

Please see the data_overview.xlsx, the paper and its supporting information.