Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

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.