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Updated: Sep 8, 2025

The Floating Lab: Standard Operational Procedure for Collecting and Filtering Seawater Samples from Operating Ferries for Environmental DNA Analysis
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Do pseudogenes pose a problem for metabarcoding marine animal communities?

Jessica A Schultz1,2, Paul D N Hebert1,2

  • 1Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada.

Molecular Ecology Resources
|June 14, 2022
PubMed
Summary
This summary is machine-generated.

Nuclear mitochondrial pseudogenes (NUMTs) can inflate DNA metabarcoding diversity. This study found NUMTs pose minimal risk to biodiversity assessments when using longer amplicons and comprehensive databases, ensuring accurate species identification.

Keywords:
COIDNA barcodingNUMTshigh-throughput sequencingmarine biodiversitynuclear mitochondrial pseudogenes

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Area of Science:

  • Molecular Ecology
  • Genomics
  • Bioinformatics

Background:

  • DNA metabarcoding relies on mitochondrial sequence variation for species identification.
  • Nuclear mitochondrial pseudogenes (NUMTs) are non-functional copies of mitochondrial DNA that can be amplified alongside genuine mitochondrial DNA.
  • NUMTs can lead to inflated estimates of species diversity and genetic variation in metabarcoding studies.

Purpose of the Study:

  • To quantify the incidence and characteristics of NUMTs in marine animal genomes.
  • To assess the impact of NUMTs on biodiversity estimates derived from DNA metabarcoding.
  • To evaluate strategies for mitigating NUMT-induced inflation in species counts and genetic variation.

Main Methods:

  • Analysis of 156 marine animal genomes for NUMTs within the cytochrome c oxidase I (COI) barcode region.
  • Identification of NUMTs based on the presence of indels or stop codons.
  • Simulation of metabarcoding scenarios using varying amplicon lengths and positions to assess NUMT impact.

Main Results:

  • 309 NUMTs (≥150 bp) were detected across 156 species, averaging 1.98 per species.
  • 75% of detected NUMTs lacked characteristic signs like indels or stop codons, making them difficult to distinguish from functional sequences.
  • NUMTs can inflate operational taxonomic unit (OTU) counts by up to 21% and intraspecific variation by 15% with standard amplicon lengths (313 bp).

Conclusions:

  • NUMTs pose a significant challenge to accurate biodiversity assessments, particularly with short amplicons common in environmental DNA studies.
  • Employing longer amplicons and considering NUMT position can substantially reduce inflation in OTU counts and barcode variation.
  • Developing comprehensive reference databases including both mitochondrial sequences and their NUMT derivatives is crucial for accurate metabarcoding.