The Difference Between Shotgun Metagenomics and 16S rRNA Sequencing

This is a widely used method for identifying and comparing bacteria within a given sample. The 16S rRNA gene is highly conserved across different bacterial species, allowing researchers to identify bacteria by sequencing this specific gene region.

• Cost-effective: Less expensive compared to other sequencing methods.
• Widely used: A well-established technique with extensive reference databases.
• Quick: Provides rapid insights into the bacterial composition of a sample.

• Limited scope: Only targets bacteria, missing out on other microorganisms like viruses, fungi, and archaea.
• Resolution limitations: May not distinguish between closely related species or strains.
• Bias: Prone to amplification bias, potentially skewing results.

PCR is a molecular biology technique used to amplify specific DNA sequences, making it easier to detect and quantify these sequences. In microbiome studies, PCR can target specific genes, including those from bacteria, viruses or other microorganisms.  However, because PCR must specify in advance which DNA sequences to amplify, this method only detects a small, limited number of pre-specified organisms rather than identifying the entire microbiome. 

• Highly specific: Can target and amplify specific DNA sequences with great precision.
• Fast and efficient: Provides results quickly and can detect even low-abundance organisms.
• Versatile: Can be applied to a wide range of genetic targets.

• Targeted approach: Only amplifies the sequences of interest, missing all other important genetic material. In practice, PCR methods only identify 20-30 common organisms rather than the hundreds of different species that live in your gut.
• Bias: Susceptible to primer bias, which can influence the detection and quantification of certain organisms.  High percentages of false positives have been observed for common PCR microbiome tests
• Limited scope: Provides only a very limited view of the entire microbial community.

Shotgun metagenomics is an advanced sequencing method that involves randomly breaking up all DNA in a sample, sequencing it, and then reassembling the sequences using computational methods. Unlike 16S rRNA gene and PCR, shotgun metagenomics captures the entire genetic material of a sample, offering a complete view of the genetic material in the microbiome.

• Comprehensive: Sequences all DNA in the sample, including bacteria, viruses, fungi, archaea and more.
• High-resolution: Provides detailed information down to the species or even strain level.
• Functional insights: Identifies genes and their potential functions, revealing the activities of the microbial community.
• Microbiome science: Increasing studies link strains and non-bacterial microbes to many aspects of health that are not captured via 16S or PCR methods, meaning that it’s only possible to fully understand the impact of the microbiome on your health with a shotgun metagenomic assessment

• Expensive: More costly due to the extensive sequencing and computational analysis required.
• Complex data: Generates extremely large amounts of data that require sophisticated computational tools to analyze.  Hundreds of different species in different amounts can be overwhelming to analyze without AI.

At Jona, we appreciate that a thorough understanding of the microbiome requires more than just identifying a few bacterial species or targeting specific genes. That's why we use shotgun metagenomic sequencing, a method that overcomes the limitations of 16S rRNA gene and surpasses the targeted approach of PCR in terms of quality, accuracy and comprehensiveness.

Our metagenomic approach sequences all DNA in a sample, providing a comprehensive view of the entire microbial community, including bacteria, viruses, fungi, protists and archaea down to a strain level. This holistic perspective allows us to understand the full complexity of the microbiome and its impact on health. Unlike 16S, which may miss subtle differences between species, or PCR, which focuses on a handful specific targets, our metagenomic sequencing provides detailed insights at the species and strain levels, as well as functional capabilities like metabolic pathways and antibiotic resistance.

However, just doing shotgun metagenomics is not enough to understand your microbiome since one could easily drown in all the data that is produced.  This is why Jona’s unique AI system decodes this data by systematically scanning all scientific studies on the microbiome to link your microbiome to every study ever published so that you can understand how your complex microbiome relates to your health and find answers. Furthermore, our AI reads all of the studies on how to change your microbiome with diet, lifestyle and supplements so that we can help you build a custom, personalized plan to change your microbiome to align with your health goals.