Labelling strategies in metabarcoding studies & how to ensure that nucleotide tags stay in place

Labelling strategies in metabarcoding studies & how to ensure that nucleotide tags stay place Metabarcoding of environmental DNA (eDNA) and extracted from bulk specimen samples is a powerful tool ecological interactions, diet biodiversity, as its labelling amplicons allows high-throughput sequencing taxonomically informative sequences many parallel. The backbone the addition sample-specific identifiers then following using these assign they originated from. This pooling hundreds thousands before thereby full utilisation capacity platforms. can be added both during PCR library preparation, i.e. when are prepared for sequencing. There three main with which achieve studies. One commonly used strategy so-called tagged approach extracts individually amplified primers carry at 5’ end. uniquely products pooled on pool amplicons. However, tag‐jumps have been documented this (Schnell et al. 2015). Tag-jumps cause switch between amplicons, resulting occurrence different than originally applied. Sequences output tag combinations not study design easily identified excluded. carrying incorrect, but already used, will incorrect assignments samples. - much detriment lead false positives artificial inflation diversity tag-jumps has led recommendations only out amplifications twin-tags cannot result increases cost workload In recently published article, we demonstrate tag-jump free single-tube preparation protocol Illumina specifically designed Tagsteady (Carøe Bohmann 2020). We circumvent two steps pools 5ʹ nucleotide-tagged had previously suggested tag-jumps; i) T4 polymerase blunt-ending end-repair step, ii) post-ligation amplification amplicon libraries. twin‐tagged investigate effect tag‐jumps. Doing this, demonstrated blunt‐ending PCR, alone or together, high proportions tag-jumps, our up ca. 49% total sequences. where were left resulted tag‐jump levels comparable background contamination study, encourage practitioners avoid pools, example by enable efficient cost-effective generation data correct assignment References Carøe C, K (2020) Tagsteady: A Molecular Ecology Resources , 20 1620–1631. Schnell IB, K, Gilbert MTP (2015) Tag jumps illuminated reducing sequence-to-sample misidentifications 15 1289–1303.