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Updated: Jun 28, 2025
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Nanopore DNA Sequencing for Metagenomic Soil Analysis
Published on: December 14, 2017
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A full-length 18S ribosomal DNA metabarcoding approach for determining protist community diversity using Nanopore
Chetan C Gaonkar1, Lisa Campbell1,2
1Department of Oceanography Texas A&M University College Station Texas USA.
Ecology and Evolution
|April 12, 2024
Summary
Full-length 18S rDNA sequencing offers higher protist taxonomic resolution than short reads in metabarcoding studies. This method improves genus identification, especially for challenging groups like dinoflagellates, enhancing ecological diversity assessments.
Area of Science:
- Eukaryotic microbiology
- Molecular ecology
- Biodiversity assessment
Background:
- DNA metabarcoding is crucial for protist diversity studies.
- Short-read sequencing of 18S rDNA markers limits taxonomic resolution.
- Full-length 18S rDNA offers greater resolution due to conserved and hypervariable regions.
Purpose of the Study:
- To develop and validate a new primer pair for full-length 18S rDNA amplification.
- To compare the efficacy of full-length 18S rDNA sequencing with short-read methods (Illumina MiSeq V4, V8-V9) for protist diversity assessment.
- To evaluate the impact of sequence length on taxonomic resolution, particularly for dinoflagellates.
Main Methods:
- Amplification of full-length 18S rDNA using a novel primer pair.
- High-throughput sequencing using Nanopore MinION (long reads) and Illumina MiSeq (short reads).
- Bioinformatic analysis to generate Amplicon Sequence Variants (ASVs) and compare taxonomic annotations.
Main Results:
- High congruency in major taxonomic group identification between long and short reads.
- Full-length 18S rDNA identified more genera (250/298, 84%) compared to V4 (226/298, 76%) and V8-V9 (213/298, 71%) short reads.
- Full-length sequencing improved resolution for dinoflagellates, detecting more genera compared to short reads, though reference database limitations persist.
Conclusions:
- Full-length 18S rDNA sequencing is a viable and effective approach for high-resolution protist diversity studies in field samples.
- This method enhances the detection of cryptic diversity, especially within challenging taxonomic groups.
- Future improvements in reference databases are needed to fully leverage the potential of long-read sequencing for protist identification.
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