Identify and compare microbes from complex microbiomes or environments

NGS-based ITS and 16S rRNA gene sequencing are culture-free ways to identify bacteria or fungi that may not be found using other methods

16S and ITS rRNA Sequencing

16S and Internal Transcribed Spacer (ITS) ribosomal RNA (rRNA) sequencing are common amplicon sequencing methods used to identify and compare bacteria or fungi present within a given sample. Next-generation sequencing (NGS)-based ITS and 16S rRNA gene sequencing are well-established methods for comparing sample phylogeny and taxonomy from complex microbiomes or environments that are difficult or impossible to study.

The prokaryotic 16S rRNA gene is approximately 1500 bp long, with nine variable regions interspersed between conserved regions. Variable regions of the 16S rRNA gene are frequently used for phylogenetic classification of genus or species in diverse microbial populations.1 The ITS1 region of the rRNA cistron is a commonly used DNA marker for identifying fungal species in metagenomic samples.2


Amplicon-based next-generation sequencing of the 16S gene offers several advantages over capillary sequencing or PCR-based approaches. Learn how it works with this guide to 16S sequencing methods.

Shotgun Metagenomics Methods Guide
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A key benefit of 16S and ITS ribosomal RNA NGS methods is that they provide a cost-effective technique to identify strains that may not be found using traditional methods. Unlike capillary sequencing or PCR-based approaches, next-generation sequencing is a culture-free method that enables analysis of the entire microbial community within a sample.  

16S rRNA NGS allows microbiologists to achieve genus-level sensitivity for metagenomic surveys of bacterial populations. ITS analysis with NGS enables rapid fungal identification to help advance our understanding of the mycobiome. Furthermore, NGS offers the ability to combine multiple samples in a sequencing run.

Scientists Discuss 16S rRNA Sequencing

NGS is Revealing the Mysterious World of Microbes
The Mysterious World of Microbes

Phil Hugenholtz, PhD explains the difference between 16S rRNA and shotgun metagenomic sequencing, and describes how NGS has made a difference in his research.

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16S Metagenomics for Bacterial Identification and Comparison

Using the 16S metagenomics workflow with the iSeq 100 System, you can achieve genus-level sensitivity for surveys of bacterial populations.

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Fungal Sequencing and Classification for Mycobiome Research

Get genus-level detection of fungi in diverse sample types with the ITS metagenomics workflow.

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Microbiology Methods Guide

All the information you need, from library preparation to final data analysis. Select the best tools for a broad range of microbiology applications for your laboratory.

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Microbiology Methods Guide
16S rRNA Sequencing Protocol
16S rRNA Sequencing Protocol

View a demonstrated protocol and FAQs for bacterial 16S rRNA amplicon sequencing, as well as example data sets from libraries generated with the protocol and run on the MiSeq System.

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ITS rRNA Sequencing Protocol
ITS rRNA Sequencing Protocol

View a demonstrated protocol for analyzing fungal or metagenomic samples that includes primer sequences and provides a recommended data analysis workflow.

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Microbes and Metagenomics Research Review

Metagenomics is one of the fastest-growing scientific disciplines. This document highlights peer-reviewed publications that apply Illumina sequencing technologies to metagenomics research.

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Microbes and Metagenomics Research Review

Illumina offers products to support NGS-based 16S and ITS rRNA analysis studies, from library preparation to data analysis and interpretation. Our user-friendly workflow can help take the guesswork out of your experiments.

Click on the below to view products for each workflow step.

Nextera XT v2 Index Kits

Multiplex samples to drive greater sample throughput.

iSeq 100 System

Affordable, fast, and accessible sequencing power for targeted or small genome sequencing in any lab.

MiSeq System

Speed, accuracy and simplicity for far-reaching applications in microbiology.

BaseSpace Apps for taxonomic classification

16S Metagenomics

Performs taxonomic classification of 16S rRNA targeted amplicon reads using an Illumina-curated version of the GreenGenes taxonomic database.

DRAGEN Metagenomics Pipeline

Performs taxonomic classification of reads and provides single sample and aggregate reporting.


The Metagenomic Phylogenetic Analysis (MetaPhlAn) tool profiles microbial community composition from metagenomic shotgun sequencing data.

One Codex

Enables metagenomics analysis by rapidly assessing your samples from BaseSpace Sequence Hub with a fast, comprehensive, accurate data platform.

Frequently Purchased Together

Shotgun Metagenomic Sequencing

This method involves comprehensively sampling all genes in all organisms present in a given complex sample. It allows microbiologists to evaluate bacterial diversity and detect the abundance of microbes in various environments.

Environmental DNA Sequencing
Environmental DNA

eDNA sequencing is an emerging method for studying biodiversity and monitoring ecosystem changes. For some sample types, using a combination of 16S or ITS sequencing with other approaches can help uncover the full breadth of diversity in an ecological sample.

16s rRNA is a subunit of a ribosome found in all bacteria and archaea. It is 1500 nucleotides long and contains nine variable regions interspersed between conserved regions.

16s rRNA sequencing is a culture-free method to identify and compare bacterial diversity from complex microbiomes or environments that are difficult to study. It is commonly used to identify bacteria present within a given sample down to the genus and/or species level. Specifically, it is an amplicon-based sequencing method that targets the 16s rRNA bacteria-specific genetic marker using a single amplicon focused on a single gene.

Because the 16s rRNA sequence is ubiquitous in bacteria and archaea, it can be used to identify a wide diversity of microbes within a single sample and single workflow. Through 16s rRNA sequencing, one can identify taxa present in a sample. This leads to a greater understanding of our microbial communities and their interactions with us.

Both the ribosome and its subunits are characterized by their sedimentation coefficients, expressed in Svedberg units (symbol: S). In this case, 16s means it takes 16 Svedberg units of time for the ribosome to sediment in a solution.

All bacteria and archaea have a 16s rRNA sequence.

In amplicon sequencing of 16S rRNA, the primers used bind within regions that are not 100% conserved across bacteria. This leads to some regions of certain bacteria not being included in sequencing. Additionally, chloroplasts have some homology to 16s rRNA genes and may amplify. Because microbiome samples can come from a wide variety of sources with variable compositions, it’s recommended to use control samples when investigating new sample types.

16S DNA refers to the gene in the bacterial genome that codes for the 16S rRNA. 16S rRNA is the rRNA that is transcribed from the 16S DNA gene. The Illumina 16S Metagenomic Sequencing Library Preparation protocol uses DNA as input, and the PCR primers target the variable regions V3 and V4 of the 16S DNA gene for the amplicon PCR.

The 16S Demonstrated Protocol provides an option for creating Illumina compatible libraries from the target of your choice. Fungi and other organisms do not have 16s rRNA genes, however, they have other conserved regions such as 18S and ITS regions. Any amplicon can be used to do similar diversity analysis studies.

The internal transcribed spacer 1 (ITS1) region of the rRNA cistron is a commonly used DNA marker for identification of fungal species in metagenomic sample. ⁵

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MiSeq System
MiSeq System

The MiSeq benchtop sequencer enables targeted and microbial genome applications, with high-quality sequencing, simple data analysis, and cloud storage.

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Nextera XT and Nextera DNA Flex
Nextera XT and Nextera DNA Flex

Prepare sequencing libraries for small genomes, amplicons, plasmids, and other applications.

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MiSeq, 16S rRNA Sequencing and the American Gut Project
16S rRNA Sequencing and the American Gut Project

Illumina NGS is enabling "citizen science" metagenomics studies of the human microbiome.

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16S rRNA Sequencing Webinar
16S rRNA Sequencing Workflow Video

A detailed look at our demonstrated 16S rRNA analysis workflow, including data visualization and success stories.

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  1. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991;173(2):697–703
  2. Schoch CL, Seifert KA, Huhndorf S, et al. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci. 2012;109(16):6241-6
  3. The Human Microbiome Project Consortium (2012) Structure, function and diversity of the healthy human microbiome. Nature 486:207–14.
  4. McCafferty J, Mühlbauer M, Gharaibeh RZ, Arthur JC, Perez-Chanona E., et al. (2013) Stochastic changes over time and not founder effects drive cage effects in microbial community assembly in a mouse model. ISME Journal doi: 10.1038/ismej.2013.106.
  5. Schoch CL, Seifert KA, Huhndorf S, et al. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci. 2012;109(16):6241-6.