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Tag jumps illuminated--reducing sequence-to-sample misidentifications in metabarcoding studies.

Ida Baerholm Schnell1,2, Kristine Bohmann1,3, M Thomas P Gilbert1,4

  • 1Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, 1350, Copenhagen K, Denmark.

Molecular Ecology Resources
|March 6, 2015
PubMed
Summary
This summary is machine-generated.

Tag jumping, or false tag combinations, affects Illumina metabarcoding accuracy. This study quantifies tag jumping in environmental DNA (eDNA) studies, finding it impacts 2-3% of sequences and can skew ecological diversity results.

Keywords:
chimerasdiversity assessmentenvironmental DNAmetabarcodingsecond-generation sequencingtag jumping

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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
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Area of Science:

  • Molecular Ecology
  • Environmental DNA (eDNA) Metabarcoding
  • Bioinformatics

Background:

  • Metabarcoding using second-generation sequencing is crucial for ecological studies.
  • Accurate sample origin tracking relies on unique sequence tags.
  • Tag jumping, or false tag combinations, can compromise data integrity.

Purpose of the Study:

  • To investigate and quantify tag jumping in Illumina-based metabarcoding.
  • To identify potential sources of tag jumping during library preparation.
  • To propose strategies for mitigating tag jumping in ecological research.

Main Methods:

  • Applied tagged generic arthropod and mammal primers to mixed-template DNA extracts from bat guano and leech gut contents.
  • Analyzed sequencing outputs to identify sequences with incorrect tag combinations (tag jumping).
  • Quantified the prevalence of tag jumping in both bat and leech diet studies.

Main Results:

  • An average of 2.6% of sequences in the leech diet study exhibited tag jumping.
  • An average of 2.1% of sequences in the bat diet study exhibited tag jumping.
  • Tag jumping can arise from blunt-ending of amplicons and chimera formation during library PCR.

Conclusions:

  • Tag jumping poses a significant challenge to accurate sample assignment in Illumina metabarcoding.
  • False sequence assignments due to tag jumping can artificially inflate diversity estimates.
  • Implementing specific measures is essential to avoid tag jumping and ensure reliable eDNA data.