What Is Microbiome NGS and How Does It Work?

A microbiome is a dynamic community of microorganisms, including bacteria, viruses, and fungi, that inhabit a particular environment. These complex ecosystems are found everywhere, from the soil beneath our feet to the depths of the ocean. The human body itself is a host to numerous microbiomes, with the gut containing a particularly dense and influential population of microbes.

Next-Generation Sequencing, or NGS, is a technology that has transformed the study of genetics. It allows for the rapid and simultaneous sequencing of millions of DNA or RNA fragments, providing a massive amount of genetic information quickly. This high-throughput capability means scientists can determine the precise order of nucleotides within a DNA molecule on an unprecedented scale. Applying this sequencing technology to microbial communities created the field of microbiome NGS.

Key Approaches in Microbiome Sequencing

The study of microbiomes through NGS primarily relies on two distinct methods. The first is 16S rRNA gene sequencing, which acts like a census for bacteria. This technique focuses on a single gene, the 16S ribosomal RNA gene, which is present in all bacteria but contains variable regions that differ between species. By sequencing this specific gene, researchers can identify which types of bacteria are present and their relative proportions.

This targeted approach is effective for creating a taxonomic snapshot of the community. The process involves using specific primers to amplify only the 16S rRNA gene from the total DNA extracted from a sample. This method provides a cost-effective way to survey bacterial diversity across a large number of samples, making it a popular choice for initial explorations.

A more comprehensive method is shotgun metagenomic sequencing, which provides a deeper look into the microbial community’s functional capabilities. Instead of targeting a single gene, this approach sequences all the genetic material from every microorganism in the sample. This technique reveals not only who is present but also what they have the potential to do by identifying all the genes within the community.

Shotgun sequencing generates a vast dataset that includes genes involved in metabolism, antibiotic resistance, and the production of various compounds. While more expensive and computationally intensive than 16S sequencing, it offers a detailed functional profile of the microbiome. This links community composition to its metabolic output and its influence on the surrounding environment.

The Microbiome NGS Process

The journey from a physical sample to a detailed digital map of a microbiome involves a multi-step process that combines laboratory work with data analysis. It begins with careful sample collection, which can range from a soil core to a stool sample. The method of collection is designed to preserve the microbial community and prevent contamination or degradation of the genetic material.

Once a sample arrives at the lab, the first major step is DNA extraction. This procedure involves breaking open the tough cell walls of the various microbes to release their DNA into a solution. The goal is to efficiently isolate high-quality DNA from the wide array of microorganisms present.

Following extraction, the DNA is prepared for sequencing in a step called library preparation. The long strands of DNA are fragmented into shorter, more manageable pieces. Special adapters, which are small known DNA sequences, are then attached to the ends of these fragments to allow them to bind to the sequencing flow cell.

The prepared library is then loaded into a Next-Generation Sequencing machine. Inside the sequencer, a process called sequencing by synthesis occurs. The machine reads the millions of DNA fragments in parallel, determining the sequence of nucleotide bases (A, C, G, and T) for each one. This step generates a raw data file containing millions of short DNA sequences, called “reads.”

The final stage is bioinformatic analysis, which uses specialized software and computing power to make sense of the raw sequencing data. The short reads are checked for quality and then compared against reference databases of known microbial genomes. This analysis identifies the types of microbes present, their abundance, and the specific genes and functional pathways encoded in their DNA.

Revealing Microbial Worlds with NGS Data

In human health, NGS has linked specific imbalances in the gut microbiome, known as dysbiosis, to a range of conditions. For example, distinct microbial profiles have been associated with inflammatory bowel disease (IBD) and obesity. This technology has also revealed connections to mental health through the gut-brain axis.

Beyond human health, NGS is a tool in environmental science. It is used to analyze the health of agricultural soil by characterizing microbial communities that support plant growth. In aquatic environments, sequencing the microbiome of water samples helps monitor for pollutants and harmful algal blooms. This technology has also led to the discovery of novel microorganisms in extreme environments.

The information from NGS is also advancing personalized medicine. By analyzing an individual’s gut microbiome, clinicians can develop personalized nutrition plans to optimize microbial function. This data is also guiding the development of new diagnostic tools, next-generation probiotics, and therapies designed to restore a healthy microbial balance.

Why NGS Revolutionized Microbiology

Next-Generation Sequencing fundamentally changed microbiology by overcoming a major limitation of traditional methods. For over a century, the primary way to study bacteria was through microbial culturing, which involves growing microbes on a petri dish in a lab. This approach presented a skewed and incomplete picture of microbial ecosystems, a phenomenon known as “The Great Plate Count Anomaly.”

This anomaly refers to the massive discrepancy between the number of cells observed directly in a sample and the number that could actually be grown in culture. It is estimated that over 99% of all microbial species cannot be cultivated using standard laboratory techniques. As a result, the vast majority of microbial life remained invisible to science. This inability to culture most microbes meant our understanding was heavily biased toward the small fraction of organisms that could survive in a lab setting.

NGS provided the solution by being a “culture-independent” method. By directly sequencing DNA from an environmental sample, researchers could bypass the need to grow organisms in the lab altogether. This allowed them to access the genetic information of the entire community, including the unculturable majority. This shift from culture-based to sequence-based analysis sparked a revolution, unveiling the true complexity of the microbial world.