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The use of taxon-specific reference databases compromises metagenomic classification.

Vanessa R Marcelino1,2,3, Edward C Holmes4,5, Tania C Sorrell4,6

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Using taxon-specific databases for fungal DNA analysis can lead to false positives. A broader, kingdom-agnostic approach is recommended for accurate microbiome profiling.

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Area of Science:

  • Microbiology
  • Bioinformatics
  • Genomics

Background:

  • Metagenomic studies often rely on bioinformatics tools to classify microbial communities.
  • The HumanMycobiomeScan tool was developed to identify fungal taxa in metagenomic samples.
  • Previous analyses using this tool characterized the human gut mycobiome of different populations.

Purpose of the Study:

  • To evaluate the accuracy of taxon-specific reference databases in metagenomic classification.
  • To demonstrate the potential for false-positive identifications using such databases.
  • To propose a more robust approach for analyzing microbial eukaryotes in metagenomic data.

Main Methods:

  • Re-analysis of a case study using a reference database containing amphibian and reptile genomes.
  • Comparison of results from a fungal-only database versus a kingdom-agnostic database.
  • Application of sequence read mapping against curated reference genomes.

Main Results:

  • A fungal-specific database incorrectly identified herpetological taxa (turtles, frogs, snakes) as abundant in the human gut.
  • Re-analysis with a kingdom-agnostic pipeline revealed a broader range of microbial eukaryotes but fewer specific fungal families.
  • The study identified significant limitations in using taxon-specific databases for metagenomic classification.

Conclusions:

  • Taxon-specific reference databases can yield high false-positive rates in metagenomic analysis.
  • The use of curated, comprehensive databases encompassing all domains of life is crucial for accurate species profiling.
  • This highlights the importance of selecting appropriate bioinformatics pipelines for microbiome research, especially for microbial eukaryotes.