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rCRUX: A Rapid and Versatile Tool for Generating Metabarcoding Reference libraries in R.

Emily E Curd1, Luna Gal2,3, Ramon Gallego4

  • 1Vermont Biomedical Research Network, University of Vermont, VT, USA.

Environmental DNA (Hoboken, N.J.)
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Summary
This summary is machine-generated.

The rCRUX R package generates comprehensive DNA barcode reference libraries for metabarcoding. It overcomes limitations of existing tools by using sequence homology and primer compatibility for accurate taxonomic classification.

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Area of Science:

  • Bioinformatics
  • Molecular Ecology
  • Genomics

Background:

  • Accurate taxonomic classification of DNA sequences is crucial for biodiversity monitoring and research.
  • Existing reference barcode databases face challenges in curation due to rapidly growing sequence data and the need for diverse gene regions and taxa.
  • There is a need for user-friendly computational tools to generate custom metabarcoding reference libraries.

Purpose of the Study:

  • To present rCRUX, an R package designed to automate the generation of comprehensive, curated metabarcoding reference libraries.
  • To provide a tool that relies on sequence homology and PCR primer compatibility, avoiding metadata limitations.
  • To facilitate accurate taxonomic classification for diverse DNA sequencing applications.

Main Methods:

  • rCRUX simulates in silico PCR to identify seed amplicons based on user-defined primer sequences.
  • Iterative BLAST searches against the NCBI nt database are performed using stratified random sampling.
  • Databases are dereplicated and cleaned to ensure taxonomic accuracy and lowest common agreement.

Main Results:

  • rCRUX generated more comprehensive reference databases for several loci (MiFish 12S, Taberlet trnl, fungal ITS, Leray CO1) compared to existing tools (CRABS, MetaCurator, RESCRIPt, ecoPCR).
  • The package successfully generated 24 reference databases for 20 metabarcoding loci, including those lacking dedicated curation efforts.
  • Demonstrated the utility of rCRUX in creating bespoke reference libraries for various taxonomic groups and gene regions.

Conclusions:

  • rCRUX is an effective and user-friendly tool for generating curated, comprehensive DNA barcode reference databases for custom loci.
  • The package enhances the accuracy and efficiency of taxonomic classification in metabarcoding and DNA sequencing studies.
  • rCRUX addresses the growing need for specialized reference libraries in biodiversity research and monitoring.