April 11, 2025
The global next-generation sequencing market was valued at $13 billion in 2023 and is expected to grow to $97.8 billion by 2035, with a compound annual growth rate (CAGR) of 18.3% from 2024 to 2035. The sequencing market is increasing due to the rising incidence of genetic disorders and cancer. There are many methods, innovations, and technological developments taking place that are expanding this market.
One such approach is 16S ribosomal RNA sequencing, a targeted sequencing technique used to identify, do detail analysis and compare bacterial species in a sample by targeting the highly conserved 16S rRNA gene. This method is typically performed using next-generation sequencing (NGS) or Sanger sequencing.
Traditional bacterial identification based on phenotypic characteristics often lacks the accuracy provided by genotypic methods. This is why 16S rRNA gene sequencing is preferred for species identification, as it offers more accurate and precise identification.
In this article, you will uncover the core of 16S rRNA sequencing for species identification, its methodology, limitations, application, and more. Let’s explore it below!
Source: ResearchGate
Do you know why and when the 16S rRNA gene took place? In the 1980s, researchers such as Carl Woese demonstrated that phylogenetic relationships among bacteria could be determined by comparing the sequences of the 16S rRNA gene, a stable part of the genetic code. This approach provided a more accurate and standardized method for identifying bacterial species compared to traditional phenotypic methods.
This led to identifying the 16S rRNA gene as a valuable tool for taxonomic purposes. The 16S rRNA gene, also known as 16S rDNA, became the standard for bacterial identification due to its universality across prokaryotes.
The 16S rRNA gene is a huge contributor to the structure and function of the ribosome, facilitating protein synthesis in prokaryotic organisms. Its stability and conservation across different bacterial species make it a reliable marker for identifying and differentiating species.
The 16S rRNA gene serves as a molecular marker specifically for prokaryotes. This gene is present in all bacterial species and archaea, which is a great medium through which researchers get to identify microbial organisms based on genetic information, overcoming the limitations of traditional phenotypic methods.
The 16S rRNA gene contains both conserved and variable regions. The conserved regions are important for universal primer binding, while the variable regions help differentiate between closely related bacterial species. This dual nature of the gene allows for both broad identification across all prokaryotes and the fine-tuning needed to distinguish specific species.
To better understand the topic, you must explore the core of 16-s ribosomal RNA sequencing identification below and uncover the methodologies of this technology.
Source: NIH
This section will reveal the procedure or methodologies you need to follow for species identification from the 16S rRNA gene sequencing. By combining these methods, 16S rRNA gene sequencing provides powerful insights into microbial communities, their diversity, and their potential role in health and disease. Let’s explore them below!
To start this procedure, you need to collect skin microbes using noninvasive methods such as swabs or more invasive techniques like punch biopsies. Each method has varying levels of penetration and results in slightly different microbial profiles, so you must maintain consistency in sampling and collecting negative controls. Skin microbiome studies require careful handling to ensure accurate and representative sampling.
DNA extraction is a critical step in 16S rRNA gene sequencing, as the quality and purity of the extracted DNA directly affect the accuracy and reliability of the sequencing results. The cell lysis (breaking down the cell membrane and/or cell) step is crucial, as bacteria have different susceptibilities to lysis. Breaking down is particularly useful for tough or difficult-to-lyse microbes, such as Gram-positive bacteria. Common methods for cell lysis include chemical treatments (detergents, enzymes) and physical methods (bead beating) to ensure the effective breakdown of the cells.
Once DNA is isolated, it is PCR-amplified using primers that target the 16S rRNA gene. These primers are chosen to amplify specific gene regions, which contain both conserved and variable regions. The conserved areas allow for broad amplification across all bacterial species, while the variable regions, such as V1-V3, V3-V5, and V4-V5, provide taxonomic information useful for differentiating bacterial species. For example, the V1-V3 region is commonly used in skin microbiome studies to distinguish among species in the Staphylococcus genus.
After PCR amplification, the next step is to sequence the amplified products. Here comes the common next-generation sequencing (NGS) platforms for 16S rRNA gene analysis, including Illumina MiSeq, Roche 454 (which is being phased out), and Thermo Fisher Ion Torrent. These are platforms that use different sequencing technologies, such as sequencing during synthesis and massively parallel sequencing. These platforms provide high-throughput sequencing, enabling the generation of millions of reads.
Following sequencing, bioinformatic tools like Mothur and QIIME are used to interpret the data for community comparisons, diversity analysis, and taxonomy assignment. The Operational Taxonomic Unit (OTU), which classifies sequences with high similarity (usually >97%) into a single category, is a widely used idea in 16S rRNA gene sequencing.
Data can be analyzed using two approaches:
As you have covered the methodologies or procedures to identify species using the 16S rRNA gene sequencing method, below, you explore the application of the 16S rRNA gene sequencing.
The applications below show just how deep and wide the influence of 16S rRNA sequencing is in different industries. Let’s explore each application and consider real-world examples.
In food microbiology, 16S rRNA sequencing detects bacterial contamination, including harmful pathogens like Salmonella or E. coli. The method helps identify not only harmful species but also other microbes present that may be overlooked in traditional microbiological testing. For fermented foods like yogurt, beer, or cheese, 16S rRNA sequencing identifies the strains of beneficial microbes involved in fermentation. This helps ensure consistency in the production process and the quality of the final product.
For example, a study applied high-throughput 16S rRNA sequencing to analyze bacterial communities in ready-to-eat (RTE) salads across Europe. Despite traditional culture-based methods failing to detect foodborne pathogens like Salmonella and E. coli, the sequencing method revealed low abundance but metabolically active opportunistic pathogens, including Aeromonas hydrophila and Rahnella aquatilis.
The use of 16S rRNA gene sequencing was crucial in distinguishing B. hinzii from B. avium. This method contributed hugely compared to traditional technology by providing definitive species-level identification, which was essential for accurate diagnosis and treatment in this atypical clinical case. In molecular diagnostics, 16S rRNA sequencing offers a critical advantage in cases where traditional biochemical methods fail, especially when dealing with rare or novel pathogens.
For example, a 69-year-old male patient developed severe septicemia following a 3-week history of jaundice and weight loss. Despite initial treatment and diagnostic efforts, the patient’s condition worsened, and he eventually succumbed to sepsis. Blood cultures revealed gram-negative rods, but biochemical tests failed to provide an accurate diagnosis. Further analysis using 16S rRNA gene sequencing identified the pathogen as Bordetella hinzii, a bacterium typically found in poultry respiratory tracts, which had never before been reported to cause human infections of such severity.
The 16S rRNA gene sequencing has become an invaluable tool for identifying bacteria. It is especially useful for identifying unusual bacterial strains or those that produce ambiguous results in biochemical profiles. In cases where a strain generates only a “low likelihood” identification through commercial systems, 16S rRNA sequencing can often provide the genus and sometimes the species identity. Over the past 20 years, 16S rRNA gene sequencing has yielded genus identification in over 90% of cases. For species identification, it identifies pathogens with an accuracy of 65% to 83%, depending on the bacterial group.
For example, 16S rRNA gene sequencing is used to analyze the diversity and structure of bacteria in the mouth, helping researchers understand oral health and disease. This technology is also crucial in studying how changes in the oral microbiome relate to systemic conditions like diabetes, heart disease, rheumatoid arthritis, Alzheimer's, respiratory problems, and pregnancy complications. The method remains popular in oral microbiology due to its quick processing, ease of analysis, and cost-effectiveness.
16S rRNA sequencing has emerged as a powerful tool for enhancing water quality monitoring, particularly in identifying microbial communities and tracing sources of contamination. Unlike traditional methods that only detect fecal indicator bacteria (FIBs), 16S rRNA sequencing provides a more detailed and accurate profile of bacterial populations in water samples. This DNA-based technique enables the detection of a wide range of microorganisms, including pathogens and other waterborne contaminants. It helps pinpoint specific sources of fecal pollution, such as septic tank leaks or agricultural runoff.
For example, a study focused on the application of high-throughput 16S rRNA sequencing to identify sources of fecal contamination in groundwater used for drinking in rural Wainfleet, Ontario, Canada. The area has been under a long-standing boil water advisory due to persistent fecal indicator bacteria (FIB) contamination in private wells. The study aimed to enhance traditional water quality testing methods and pinpoint specific sources of fecal pollution using DNA-based tools such as quantitative PCR (qPCR) and 16S rRNA gene sequencing.
The integration of 16S rRNA gene sequencing into forensic science has opened new possibilities for body fluid identification, particularly in distinguishing saliva from other bodily fluids in criminal investigations. Traditionally, saliva identification relied on the α-amylase test, which lacked specificity and was prone to false positives.
For example, a study utilized 16S rRNA sequencing to analyze the microbial communities in saliva samples from five different regions, focusing on the V3-V4 hypervariable regions of the 16S rRNA gene. The results revealed a distinct salivary microbiome dominated by species like Streptococcus, Neisseria, and Rothia, with variations influenced by geographical location. This research suggests that salivary microbial profiles could serve as a reliable marker for body fluid identification and potentially for biogeographic inference, offering a promising advancement in forensic applications.
These are some of the major examples that show the contribution and importance of 16S rRNA sequencing technology. However, as you have to uncover the several usages, it comes with several limitations that need to be discussed. Let’s explore them all below!
While 16S ribosomal RNA sequencing offers many advantages, it also has some limitations that need to be addressed. Its limitations—particularly regarding species-level resolution, database completeness, and the ability to differentiate closely related taxa—highlight the need for ongoing improvements in sequencing technology. Let’s explore them in depth below!
16S sequencing currently struggles to differentiate species within closely related bacterial groups. For example, it cannot distinguish between species within the Mycobacterium tuberculosis complex or the M. avium and M. chelonae complexes, which reduces the accuracy of identifying pathogens at the species level.
Although extensive, publicly available databases like GenBank often lack proper curation. This can lead to issues such as misidentifications or an inability to identify novel pathogens that have not been sequenced or cataloged. To enhance accuracy, the databases must be continuously updated to include a wider range of sequences from rare or emerging pathogens.
Many bacteria have very similar 16S rRNA sequences, making it difficult to distinguish between them at the genus and species levels. This issue is particularly problematic for bacteria within the same genus or closely related species that share high sequence homology.
Some bacterial genomes contain multiple copies of the 16S gene, which may differ slightly from one another. These variations can complicate the sequencing process, potentially leading to inaccurate or inconsistent results in species identification.
Uncovering these insights helps you understand the challenges. New advanced platforms like Biostate AI have managed to overcome the limitations by providing powerful solutions, ensuring accurate species identification, precise microbial profiling, and better differentiation of closely related bacteria—all at an affordable price.
However, this particular approach is also improving, and several future projects and developments are planned. Let's explore them below.
Do you know if Illumina has introduced a gene-sequencing device? Illumina MiSeq i100 Series: Illumina has introduced compact, cost-effective benchtop sequencers, such as the MiSeq i100, priced at $49,000, and the i100 Plus, priced at $109,000. These devices deliver results in just four hours, making sequencing more accessible to smaller research and testing laboratories.
The continuous advancement of 16S rRNA gene sequencing technology makes it more affordable and user-friendly. Thus, 16S rRNA sequencing is becoming more accessible to animal scientists, allowing researchers to investigate the role of microbiomes in animal health and productivity.
Future advancements may integrate microarray-based technologies with 16S rRNA sequencing or other housekeeping gene targets. This combination could enhance sensitivity and provide more reliable platforms for identifying microbial species.
While intragenomic 16S sequence variation complicates community analysis, it can enhance strain-level discrimination. When properly accounted for, multiple polymorphic 16S copies enable more precise differentiation of closely related groups. Longer reads and advanced sequencing platforms will improve the ability to distinguish closely related bacterial strains, moving beyond species-level identification.
Combining 16S rRNA data with metagenomics, transcriptomics, and proteomics will provide transparency on how microbes function and interact in different environments. Improved algorithms will make taxonomic classification more accurate, handle intragenomic variations better, and enhance microbial diversity analysis.
With the advancement in the 16S rRNA sequencing field, a major launch took place, where Microbiome Insights launched long-read sequencing services that generate longer sequences (up to 4 kb), enhancing the analysis of microbial communities. This service includes comprehensive workflows from experimental design to bioinformatics analysis. This technology was also recognized as "Method of the Year in 2022" by Nature Methods.
Machine learning is integrated into 16S rRNA gene sequencing by analyzing the generated sequence data to identify and classify bacterial species with greater accuracy, particularly when dealing with complex microbiome samples. Algorithms extract patterns and relationships within the data that might not be readily apparent through traditional methods, allowing for better prediction.
For example, Companies like Illumina are integrating artificial intelligence (AI) with sequencing technologies. Illumina's partnership with NVIDIA aims to enhance genomic data analysis by leveraging AI, potentially leading to more efficient and accurate sequencing workflows.
As you've explored the core principles, methodologies, applications, and limitations of 16S rRNA gene sequencing, it's clear that while this technique remains a powerful tool for microbial identification, ongoing advancements are shaping its future.
16S ribosomal RNA sequencing has contributed hugely to the identification of species by enabling precise classification of bacteria and archaea, including those previously unculturable. It plays an important role in microbial taxonomy and continues to expand as advancements in sequencing technology and bioinformatics refine classification methods and uncover new species.
Beyond taxonomy, this technique remains essential in medical diagnostics, environmental monitoring, and biotechnology. AI-driven platforms like Biostate.ai are enhancing sequence analysis, improving taxonomic classification, and uncovering novel microbial interactions. Biostate.ai is a space that offers a complete solution for RNA sequencing of any sample and does everything, from handling sample collection to final insights, at an affordable rate. Get Your Quote Now!
