Next Generation 16S rRNA Sequencing

About Next Generation 16S Sequencing

Many sites of the human body are colonized by complex communities of microbes (the "human microbiome") in both health and various disease states. Highly diverse, polymicrobial specimens are often difficult, or even impossible, to fully characterize by techniques in common clinical use:

Figure 1. Examples of conventional 16S rRNA gene sequencing results from a bacterial isolate and a polymicrobial specimen. For the bacterial isolate (top), Sanger sequence data produces a clean electropherogram that can be used to provide a species-level taxonomic classification. For the polymicrobial sample (bottom), Sanger sequencing generates a different electropherogram for each species present, resulting in mixed signal which is uninterpretable.
  • Culture-based identification relies upon the ability of organisms to grow and replicate in vitro. Therefore, detection of fastidious or slow-growing organisms, or those rendered inviable due to processing (such as in formalin-fixed paraffin embedded tissue specimens) or during storage (such as anaerobes which have been exposed to oxygen) is limited. Furthermore, only a limited number of species can be practically classified by this approach.
  • Molecular methods, such as our 16S rRNA gene sequencing assay, do not require prior culture and have improved ability to identify inviable or fastidious organisms from direct specimens. However, in a polymicrobial speciemen only the predominant organism may be identified, or an uninterpretable signal may be generated (Figure 1).

Figure 2. High-powered magnification of a next-generation sequencing run. Each fluorescent spot represents an individual DNA molecule undergoing sequencing. The color of the spot indicates the identity of the nucleotide being interrogated during the current sequencing cycle. Image from Shendure, Porreca et al. Science (2005).

In contrast to conventional approaches, next-generation DNA sequencing (alternatively termed "NGS", "high-throughput sequencing", "massively parallel sequencing", or "deep sequencing") provides independent sequence data from millions of individual DNA molecules (Figure 2), allowing each fragment to be classified independently.

This unique ability extends upon the advantages of current molecular methods by allowing us to catalog the organisms present within even very complex polymicrobial bacterial communities, directly from patient specimens.

Information on Available Assays

Our lab currently offers high-fidelity Illumina next-generation DNA sequencing of clinical specimens which contain multiple bacterial DNA templates. Methods are validated for the purpose of clinical molecular diagnosis and patient care. Research services are also available - please contact us for additional information.

This test is available as reflex testing for specimens which are expected to be polymicrobial based on broad range bacterial PCR.

To learn more about clinical applications and methodology, please see the following publications from our lab:

Contact for details or questions.

Clinical Reporting

Upon completion of testing, a report is issued describing the results of 16S next generation sequencing. To view a sample report, click here or the thumbnail at left.

For additional information on how to submit a request and recieve a report, please contact us!