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

Abstract

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, 1/20 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.

An appetite for pests: Synanthropic insectivorous bats exploit cotton pest irruptions and consume various deleterious arthropods

“…PCR products were assigned forward and reverse nucleotide tags yielding a unique combination of 5’ nucleotide tags (7–8 nucleotides long) allowing pooling of DNA amplicons with the ability to trace back sequences to their samples according to their tags (Binladen et al, 2007). PCR amplifications were performed with matching tags (e.g., F1‐R1, F2‐R2, etc) to ensure that tag jumps would not cause false assignments of sequences to samples (Carøe & Bohmann, 2020; Schnell, Bohmann, & Gilbert, 2015). All sample DNA extracts were amplified in three PCR replicates, each with a unique matching tag combination to allow tractability and to aid in the identification of erroneous sequences by filtering out sequences that only appeared in one replicate (Alberdi et al, 2018).…”

eDNA‐based biomonitoring at an experimental German vineyard to characterize how management regimes shape ecosystem diversity

“…PCR products were visualized using gel electrophoresis (GE). PCR replicates were pooled into a single pool based on gel intensity, then converted to Illumina sequencing libraries using the TagSteady PCR‐free library protocol (Carøe & Bohmann, 2020) that minimizes pooling bias and tag jumping between amplicons (Schnell et al., 2015). Library quality was assessed using NEBNext Library Quant Kit Quick Protocol (NEB) following the manufacturer's standard protocol (Feld, Nielsen, Hansen, Aamand, & Albers, 2016).…”

The role of the gut microbiota in the dietary niche expansion of fishing bats

“…All amplicon pools were subsequently purified at 1:1 beads:DNA volumeratio to remove non-target DNA and primer dimers using SPRI beads [69,70]. Sequencing libraries were prepared using the Tagsteady library building protocol [71], and sequenced on an Illumina MiSeq machine using 250PE chemistry.…”