TY - JOUR

T1 - Tagsteady

T2 - A metabarcoding library preparation protocol to avoid false assignment of sequences to samples

AU - Carøe, Christian

AU - Bohmann, Kristine

PY - 2020

Y1 - 2020

N2 - 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 5MODIFIER LETTER PRIME 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.

AB - 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 5MODIFIER LETTER PRIME 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.

KW - amplicon sequencing

KW - environmental DNA

KW - high-throughput sequencing

KW - tag-jumps

KW - DNA

KW - IDENTIFICATION

KW - AMPLIFICATION

KW - SPECIFICITY

KW - BIOLOGY

U2 - 10.1111/1755-0998.13227

DO - 10.1111/1755-0998.13227

M3 - Journal article

C2 - 32663358

VL - 20

SP - 1620

EP - 1631

JO - Molecular Ecology

JF - Molecular Ecology

SN - 0962-1083

IS - 6

ER -