Microbial Genomics
    • Microbial Genomics

    16S and ITS rRNA Sequencing

    Identify and compare microbes from complex microbiomes or environments using culture-free NGS-based ITS and 16S rRNA gene sequencing methods

    What are 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. These culture-free methods allow scientists to identify bacteria or fungi that may not be found using other methods.

    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 complex microbiome samples.2

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    Advantages of NGS-based 16S and ITS rRNA analysis

    A key benefit of 16S and ITS ribosomal RNA NGS methods is that they provide a cost-effective technique to identify microbes 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 diversity analysis 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.

    Applications of 16S and ITS rRNA sequencing

    Human microbiome analysis

    See how NGS can empower studies capable of surveying the genomes of entire microbial communities, including those of unculturable organisms.

    Infectious disease identification

    Learn how NGS-based methods can improve infectious disease characterization and surveillance of viruses, bacteria, fungi, and parasites.

    Demonstrated 16S and ITS rRNA protocols

    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.

    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.

    Methods for 16S RNA sequencing

    In this guide you'll learn about solutions for taxonomic profiling of bacterial communities and microbiomes. Illumina NGS workflows integrate library prep, sequencing, and data analysis.

    Recommended workflow for 16S and ITS rRNA sequencing

    1
    Prep

    Illumina DNA/RNA UD Indexes

    A fast, integrated workflow for preparing libraries for use in sequencing applications such as 16S and ITS rRNA sequencing.

    Illumina Purification Beads

    Beads for library clean-up and size selection in 30 mL, 100 mL, or 400 mL sizes. Products can be found under “Accessory products” on the Illumina DNA Prep product page. Download flyer

    2
    Sequence

    MiSeq i100 Series

    Our fastest, simplest benchtop system for 16S sequencing.

    NextSeq 1000 and 2000 Systems

    Empower discoveries with a comprehensive workflow from start to finish.

    3
    Analyze

    16S Metagenomics

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

    16S rRNA sequencing on NextSeq 1000 and NextSeq 2000 Systems

    Learn how the NextSeq 1000 and NextSeq 2000 Systems can enable efficient, high-throughput characterization capabilities of microbial populations into your laboratory.

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    Related methods

    Shotgun metagenomic sequencing

    See how this method enables microbiologists to evaluate bacterial diversity, detect the abundance of microbes in various environments, and study unculturable microorganisms that are otherwise difficult or impossible to analyze.

    Metatranscriptomics and microbial transcriptomics

    Learn about how to collectively study expressed mRNA from complex microbial communities.  This method also allows researchers to gain important insights on how microbes respond to a given environment at a specific time.

    16S rRNA sequencing FAQs

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    References

    1. Weisburg WG, Barns SM, Pelletier DA, et al. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991;173(2):697–703. doi: 10.1128/jb.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. doi: 10.1073/pnas.1117018109.