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Related Experiment Video

Updated: Dec 26, 2025

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
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Microbiota Analysis Using Sequencing by Synthesis: From Library Preparation to Sequencing.

Andrew Nelson1, Christopher J Stewart2

  • 1Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne, UK. andrew3.nelson@northumbria.ac.uk.

Methods in Molecular Biology (Clifton, N.J.)
|March 10, 2020
PubMed
Summary
This summary is machine-generated.

This protocol details a universal method for amplifying and sequencing the bacterial 16S rRNA gene

Keywords:
16S rRNADNA sequencingMiSeqMicrobiotaPCR

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Area of Science:

  • Microbiology
  • Genomics
  • Molecular Biology

Background:

  • High-throughput sequencing enables large-scale bacterial community analysis.
  • Investigating the microbiota is crucial for understanding health and disease.

Purpose of the Study:

  • To provide a protocol for universal amplification of the bacterial 16S rRNA gene's v4 region.
  • To prepare amplicons for sequencing on the Illumina MiSeq platform.

Main Methods:

  • Utilizes sequencing by synthesis (SBS) for parallel amplicon sequencing.
  • Employs universal primers for the 16S rRNA gene's v4 region.
  • Includes preparation steps for Illumina MiSeq sequencing.

Main Results:

  • Describes a method applicable to 188 genomic DNA samples.
  • Incorporates PCR positive and negative controls for validation.
  • The protocol is adaptable to various sample types for bacterial DNA detection.

Conclusions:

  • Offers a standardized method for bacterial 16S rRNA gene sequencing.
  • Facilitates comprehensive microbiota profiling across diverse sample types.
  • Enables robust investigation of bacterial communities using Illumina sequencing technology.