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

Christian Carøe¹, Kristine Bohmann¹

¹Section for Evolutionary Genomics, Globe Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Molecular Ecology Resources

|July 15, 2020

Summary

Tag-jumps in metabarcoding can inflate diversity. The new Tagsteady protocol eliminates PCR steps, preventing tag-jumps and enabling cost-effective biodiversity studies.

Keywords:

amplicon sequencing environmental DNA high-throughput sequencing tag-jumps

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Area of Science:

Molecular Biology
Ecology
Bioinformatics

Background:

Metabarcoding uses DNA sequencing to identify species in environmental samples.
Tag-jumps, a library preparation artifact, can lead to inaccurate biodiversity assessments.
Current methods to prevent tag-jumps increase costs and workload.

Purpose of the Study:

To develop a PCR-free metabarcoding library preparation protocol to minimize tag-jumps.
To evaluate the impact of specific library preparation steps on tag-jump occurrence.
To provide a cost-effective solution for accurate metabarcoding data generation.

Main Methods:

Developed Tagsteady, a PCR-free protocol for nucleotide-tagged amplicon library preparation.
Investigated the role of T4 DNA polymerase blunt-ending and postligation PCR in tag-jump formation.
Utilized twin-tagged amplicons for precise tag-jump analysis in metabarcoding.

Main Results:

T4 DNA polymerase blunt-ending and postligation PCR significantly contribute to tag-jumps (up to 49%).
The Tagsteady protocol effectively eliminates these steps, reducing tag-jumps to background levels.
Tagsteady enables efficient and cost-effective metabarcoding with minimal sequence misassignment.

Conclusions:

Tagsteady protocol offers a reliable and economical method for high-throughput metabarcoding.
Eliminating specific PCR steps is crucial for accurate biodiversity and ecological interaction studies.
This approach enhances the reliability of environmental DNA (eDNA) and bulk sample metabarcoding.