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.
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.
The Saca la Lengua project used 16S and 18S rRNA sequencing to identify the bacteria and fungi that live in the human mouth.Read Interview
Michael Bunce, PhD uses next-generation sequencing and metabarcoding methods to study environmental DNA (eDNA).Read Interview
Phil Hugenholtz, PhD explains the difference between 16S rRNA and shotgun metagenomic sequencing, and describes how NGS has made a difference in his research.Read Interview
Using the 16S metagenomics workflow with the iSeq 100 System, you can achieve genus-level sensitivity for surveys of bacterial populations.Read Application Note
Get genus-level detection of fungi in diverse sample types with the ITS metagenomics workflow.Read Application Note
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.Access Guide
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.View Protocol
View a demonstrated protocol for analyzing fungal or metagenomic samples that includes primer sequences and provides a recommended data analysis workflow.View Protocol
Metagenomics is one of the fastest-growing scientific disciplines. This document highlights peer-reviewed publications that apply Illumina sequencing technologies to metagenomics research.Access PDF
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.
BaseSpace Apps for taxonomic classification16S 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.
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.
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.