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Updated: Jun 30, 2025

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Published on: October 15, 2019

Microbial Community Profiling Protocol with Full-length 16S rRNA Sequences and Emu.

Kristen D Curry¹, Sirena Soriano², Michael G Nute¹

¹Department of Computer Science, Rice University, Houston, Texas, USA.

Current Protocols

|March 21, 2024

Summary

Full-length 16S rRNA sequencing using Oxford Nanopore Technologies (ONT) and Emu software offers precise microbial community profiling. This low-cost method enhances taxonomic classification and species-level resolution for microbiome analysis.

Keywords:

16S rRNA Oxford Nanopore Technologies bioinformatics community profile long‐read sequencing microbiome species

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Last Updated: Jun 30, 2025

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Published on: March 19, 2018

Area of Science:

Microbiology
Genomics
Bioinformatics

Background:

16S rRNA sequencing is standard for microbial community analysis.
Short-read sequencing provides partial 16S rRNA gene information.
Third-generation sequencing enables full-length 16S rRNA gene sequencing for improved accuracy.

Purpose of the Study:

To present a protocol for full-length 16S rRNA sequencing using Oxford Nanopore Technologies (ONT).
To introduce Emu software for microbial community profiling from ONT data.
To achieve species-level resolution in microbiome analysis.

Main Methods:

Generating full-length 16S rRNA sequences with ONT.
Utilizing Emu software for analyzing ONT sequences.
Building a custom reference database for Emu.

Main Results:

ONT sequencing provides complete 16S rRNA gene sequences.
Emu software overcomes limitations of incomplete databases and hardware errors.
The pipeline achieves species-level resolution for microbial communities.

Conclusions:

This protocol offers a low-cost solution for microbiome characterization.
Real-time, long-read ONT sequencing combined with Emu software enhances accuracy.
The method provides precise taxonomic classification of microbial communities.