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Comprehensive Wet-Bench and Bioinformatics Workflow for Complex Microbiota Using Oxford Nanopore Technologies
Christoph Ammer-Herrmenau1, Nina Pfisterer1, Tim van den Berg2
1Department of Gastroenterology, Gastrointestinal Oncology and Endocrinology, University Medicine Goettingen, Goettingen, Germany.
Msystems
|August 24, 2021
Summary
This study presents a standardized workflow for analyzing complex human microbiomes using Oxford Nanopore Technologies (ONT) sequencing. The optimized pipeline ensures accurate and reproducible results for buccal and rectal swabs, advancing microbiome research.
Area of Science:
- Microbiology
- Genomics
- Bioinformatics
Background:
- High-throughput sequencing, particularly Next-Generation Sequencing (NGS), has revolutionized human microbiome analysis.
- Third-generation sequencing, like Oxford Nanopore Technologies (ONT), offers rapid, long-read sequencing with high resolution.
- Current ONT protocols lack standardization for microbiome analysis, hindering clinical and translational applications.
Purpose of the Study:
- To develop and validate a comprehensive pipeline for complex microbiome analysis using ONT sequencing.
- To address the standardization challenges in sampling, storage, DNA extraction, library preparation, and bioinformatic classification for ONT.
- To provide a reliable workflow for accurate microbiome profiling from human buccal and rectal swabs.
Main Methods:
- Development of a standardized workflow encompassing sample collection, storage, DNA extraction, library preparation, and bioinformatic analysis for ONT sequencing.
- Experimental validation using buccal and rectal swabs, comparing adapted NGS protocols with optimized ONT-specific methods.
- Benchmarking of bioinformatic parameters for precision, recall, and area under the curve using 16S rRNA and metagenomic sequencing.
Main Results:
- Standard NGS protocols are not directly transferable to ONT for microbiome analysis; extended washing steps in DNA extraction are recommended.
- Both 16S rRNA and metagenomic sequencing with ONT yielded reliable and reproducible microbiome data.
- Optimized bioinformatic parameters achieved superior performance compared to existing classifiers (Kraken2, Kaiju, MetaMaps).
Conclusions:
- The presented experimental and bioinformatic pipeline enables highly accurate analysis of complex microbial structures from human buccal and rectal swabs using ONT.
- This standardized workflow facilitates rapid, reproducible, and accurate long-fragment microbiome sequencing.
- The findings support the broader application of ONT in diverse research fields for microbiome investigation.
