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Updated: Aug 9, 2026
07:33
Nanopore DNA Sequencing for Metagenomic Soil Analysis
Published on: December 14, 2017
Benchmarking Full-Length ITS Metabarcoding Across Illumina 2 × 500, PacBio, and Oxford Nanopore Sequencing Using Mock
Leho Tedersoo1, Marko Prous2,3, Meirong Chen4
1Mycology and Microbiology Center, Institute of Technology, University of Tartu, Tartu, Estonia.
Molecular Ecology Resources
|August 8, 2026
Summary
PacBio sequencing with standard pipelines offers the best results for full-length ITS metabarcoding biodiversity surveys, outperforming Illumina and Oxford Nanopore technologies. This approach minimizes errors and maximizes taxa detection for accurate soil biodiversity assessments.
Area of Science:
- Environmental DNA (eDNA) analysis
- Molecular ecology
- Bioinformatics
Background:
- Metabarcoding using standard-size DNA barcodes (> 500 bases) provides superior taxonomic resolution for biodiversity comparisons.
- Sequencing platforms and bioinformatics pipelines introduce technical biases that can significantly impact diversity estimations.
Purpose of the Study:
- To evaluate the performance of different sequencing platforms (Illumina MiSeq, PacBio Revio, Oxford Nanopore MinION) and bioinformatics pipelines for full-length ITS metabarcoding.
- To identify optimal methods for accurate soil biodiversity assessment and land use impact analysis.
Main Methods:
- Comparative analysis of Illumina MiSeq (2×500 paired-end), PacBio Revio, and Oxford Nanopore MinION sequencing platforms.
- Utilized full-length ITS amplicon sequencing data from a 103-species mock community and 45 soil samples.
- Assessed various bioinformatics pipelines, including PRONAME and Minovar, for data processing and analysis.
Main Results:
- PacBio Revio exhibited the lowest error rate and highest taxon recovery, despite initial low-quality reads.
- Illumina MiSeq showed a high proportion of chimeric and index-switched reads, with a bias towards shorter amplicons.
- MinION data, particularly with the Minovar pipeline, suffered from low-quality data and loss of rare taxa during processing.
Conclusions:
- For full-length ITS metabarcoding, PacBio sequencing combined with standard, non-ASV (amplicon sequence variant) pipelines is recommended for robust biodiversity surveys.
- Standard filtering approaches are preferable for PacBio data to retain rare taxa, while stringent ASV or OTU (Operational Taxonomic Unit) filtering is advised for Illumina to mitigate artefacts.
- Excess PCR cycles negatively impact data quality and quantitativity across all platforms; sampling design remains crucial for understanding biodiversity responses to land use.
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