16S rRNA sequencing has been used extensively in microbiome research to identify the composition of bacteria and archaea within a wide variety of microbiomes from the human gut to the Amazon rainforest.

In this guide, we will explore the principles and applications of 16S rRNA sequencing, including sample preparation, DNA extraction, library preparation, sequencing, and data analysis. We will also discuss the limitations and potential pitfalls of 16S rRNA sequencing and provide tips for optimizing your experiments.

See more about Microbiome Insights 16S rRNA sequencing service.

Table of contents

• What is 16S rRNA Sequencing Used For?
• Principles of 16S rRNA Sequencing
• Conducting 16S rRNA sequencing – Step-by-step
• Strengths and Limitations of 16S rRNA sequencing
• Important factors to consider
• 16S rRNA sequencing with Microbiome Insights
  

Resource: Are you planning a microbiome study? Download our guide: How to plan and conduct a microbiome study: 

What is 16S rRNA Sequencing Used For?

16S rRNA sequencing is research method used to identify different types of bacteria or archaea within a biological sample. For this reason, it is an extremely useful tool in microbiome research studies, however it also has a number of other applications:

Environmental microbiology: 16S rRNA sequencing can be used to identify and classify microorganisms present in soil, water, and air samples, providing insights into the diversity of microorganisms in these environments to assess pollution, contamination or microbiome profiles.

Medical microbiology: 16S rRNA sequencing can be used to identify and classify bacteria and archaea present in clinical samples, such as those collected from the human microbiome or from infected tissue. This can help diagnose and treat infections, as well as provide insights into the role of the microbiome in health and disease.

Food microbiology: 16S rRNA sequencing can also be used to identify and classify microorganisms present in food products, such as fermented foods and beverages, to ensure food safety and quality. It can also be used to screen for and identify food-borne pathogens.

Industrial microbiology: 16S rRNA sequencing can be used to identify and classify microorganisms present in industrial processes, such as those involved in the production of biotechnology products, pharmaceuticals or the treatment of wastewater.

Principles of 16S rRNA Sequencing

16s rRNA sequencing is a form of amplicon sequencing, a type of genomic sequencing that targets highly-specific regions within a genome. The 16S rRNA gene is a small, conserved gene that codes for the 16S subunit of the ribosome. The 16S rRNA gene is found in all bacteria and archaea, and is highly conserved, making it an ideal target for identifying and characterizing these microorganisms. Furthermore, there are regions of the 16S rRNA gene that are highly variable, making them useful for differentiating between individual species and strains of bacteria and archaea. Other forms of amplicon sequencing included 18S and ITS sequencing which target fungi. 

16S rRNA Sequencing Steps

Sample Preparation

As with all microbiome studies, the collection and storage of your sample is critical to obtaining accurate, reliable and reproducible 16S rRNA sequencing results. Samples can be collected in a number of ways, depending on whether they are human fecal samples, soil samples, water samples, swabs or any other type of sample. In general, the three most important factors to consider are:

1. Sterility: Sample containers must be sterile in order to prevent contamination from other microbes.

2. Temperature: In order to preserve the microbes within a sample, it is critical to freeze them. This can include storing samples in -20 or -80 freezers or snap-freezing in liquid nitrogen. Freeze-thawing is not good for microbiome results, therefore, it may be necessary to aliquot samples prior to freezing.

3. Time: Timing is essential to sample preservation. In general, it is optimal to freeze samples as quickly as possible. In some cases, it is not possible to freeze immediately, in which case temporary storage at 4 degrees is suitable or preservation buffers can be used to prolong the integrity of samples for hours to days before they are frozen.

Before DNA can be extracted from the sample, it may be necessary to pre-treat the sample to remove any contaminants or inhibitory substances that could interfere with the extraction process. This may involve adding enzymes to break down cell walls, using heat to denature proteins, or using physical methods to separate cells from debris.

DNA Extraction

Once the sample has been collected and prepared, the next step is to extract the DNA from the microorganisms in the sample. This is typically done using a DNA extraction kit, which uses a combination of chemical and physical methods to separate the DNA from other cellular components. There are a number of different DNA extraction kits available, and the choice of kit will depend on the type of sample being analyzed and the specific goals of the experiment. In general, all DNA extraction kits include the following steps:

1. Lysis: Lysis refers to the process of breaking open cells and their nuclei, in order to release their contents, including DNA. This is usually conducted by chemical processes (adding enzymes) and mechanical processes (shaking/mixing).

2. Precipitation: Once broken open, it is necessary to separate the DNA from all of the other cell contents. This is done by adding a salt solution and alcohol.

3. Purification: The isolation DNA is then washed to remove other impurities and then resuspended in a water solution.

Read our DNA extraction blog for more details about how to optimist DNA extraction for microbiome studies.

Library Preparation

Library preparation refers to the series of steps that need to be taken to prepare the DNA for sequencing. These steps include the following:

1. Amplification of the 16S rRNA gene: After the DNA has been extracted, the next step is to amplify the 16S rRNA gene using polymerase chain reaction (PCR). For this, it is necessary to use primers that specifically target one of the hypervariable regions (V1-V9) of the 16S rRNA gene. The choice of hypervariable region can influence your final results, therefore it is important to use a region that is suitable for your particular sample type.

2. Perform PCR to add molecular ‘barcodes’ to each cleaned DNA sample (if more than one sample is being sequenced): Most of the time, more than one sample is being analyzed at the same time, in which case it is necessary to add molecular barcodes to each sample to identify which reads belong to which sample after they are sequenced. 

3. Clean the DNA: In order to remove impurities that may interfere with sequencing and to remove DNA fragments that are too small or too big, it is important to clean the DNA. This is usually done by using magnetic microbeads at particular concentrations to bind the DNA at the required size but not bind other impurities. 

16S Sequencing

Once the library has been prepared, it is now ready to be sequenced. There are a number of different sequencing platforms available, including Illumina, Ion Torrent, Oxford Nanopore and PacBio. The choice of platform will depend on the specific goals of the experiment and the availability of equipment and resources. During sequencing, the DNA libraries are randomly amplified and sequenced using a combination of chemistry and optics. The resulting data generates short DNA sequences that can be aligned up to databases to identify which bacteria and archaea they belong to.

Data Analysis

Once the sequencing is complete, the final step is to analyze the data to identify and classify the microorganisms present in the sample. This is conducted using bioinformatics pipelines that help to clean up the sequencing data, by removing human reads and sequencing errors, and align the cleaned reads to public databases such as the National Center for Biotechnology Information (NCBI) database. A number of 16S sequencing pipelines are available (QIIME, MOTHUR, USEARCH-UPARSE) some of which have extensive tutorials and online user interfaces in order to support researchers without bioinformatics expertise. Once the sequencing reads have been annotated to the databases using these pipelines, various statistical approaches can be used to assess trends and associations between different samples. 

Strengths and Limitations of 16S rRNA Sequencing

While 16S rRNA sequencing is a powerful tool for identifying and characterizing microorganisms, it has some limitations in addition to strengths:

Identifying other microbes: Although 16S rRNA sequencing is very useful for identifying bacteria and archaea, it is not suitable for identifying fungi, viruses or other microorganisms in a sample. To identify these types of microbes, it would be necessary to use other forms of amplicon sequencing, qPCR or whole metagenome shotgun sequencing.

Taxonomic resolution: The confidence with which microbiome sequencing can identify genera, species and strains of microbes depends on the depth of sequencing and the databases used. In general, 16S rRNA sequencing generates lower taxonomic resolution than shotgun sequencing, meaning it is not possible to distinguish between different strains of microbial species.

Identifying microbial functions: As 16S rRNA sequencing only targets one gene on bacteria and archaea, it cannot be used to identify all of the functional capabilities of the microbes in the sample. This is only possible through shotgun sequencing. In fact, evidence from large human microbiome studies suggest that functional metagenomic data may provide more power for identifying differences between ‘healthy’ and ‘diseased’ microbiomes.

Cost: Despite generally providing less data than shotgun sequencing, 16S rRNA sequencing is much less expensive, making it more accessible and suitable for many different types of applications.

Contamination: As 16S rRNA sequencing involves the amplification of bacterial and archaeal DNA from tiny quantities, the risk of contamination from unwanted environmental bacteria is high. Therefore, it is critical to limit sources of contamination during the processing, especially in 16S rRNA sequencing studies of low microbial biomass samples.

Well-curated databases: As 16S rRNA sequencing has been used routinely for a long time in microbiome research, the databases that it uses to identify bacteria and archaea are well-curated. In certain circumstances, for example when using samples that are not well-characterized, 16S rRNA sequencing may actually identify more taxa than whole metagenome shotgun sequencing, which often relies on databases of whole microbial genomes. 

Important Factors to Consider 

To optimize your 16S rRNA sequencing experiments and obtain accurate and reliable results, there are a few key things to consider when conducting 16S rRNA sequencing for your research study or any other application:

Sample selection: 16S rRNA microbiome compositions can vary in particular samples at different times of day, in males and females and in different regions within the same organ. Therefore, it is very important to select samples that are representative of the environment or population of interest and consider the specific goals of the experiment when selecting the sample.

Follow proper sterilization and handling protocols: Proper sterilization and handling protocols are crucial for preventing sample contamination and ensuring the accuracy of the results. This is especially important with low biomass samples such as skin swabs or biopsies, which are more prone to contamination.

Use appropriate DNA extraction and library preparation kits: Choose DNA extraction and library preparation kits that are appropriate for the type of sample being analyzed and the specific goals of the experiment. DNA extraction methods can impact your 16S rRNA sequencing results.

Select appropriate sequencing coverage: The sequencing coverage, or the number and length of the reads obtained, can significantly impact the accuracy and resolution of the results. It is important to carefully consider the sequencing coverage to ensure that enough data is obtained to accurately classify the microorganisms present in the sample. This can be achieved by looking at the rarefaction curve of your sequencing results. For more complex samples, or for the accurate classification of strains, greater sequencing depth and coverage is required, however this comes at a greater financial cost.

Use positive and negative controls: Using a negative control allows you to confirm that your 16S rRNA sequencing results aren’t affected by contamination. This also allows you to account for and remove any contamination in your results, such as that coming from DNA extraction kits and laboratory reagents. Positive controls contain known combinations and quantities of microbiomes. This allows you to confirm whether your DNA extraction and library preparation protocol are suitable for identifying a variety of different organisms.

16S rRNA Sequencing with Microbiome Insights

Microbiome Insights have a team of experts available to help with your 16S rRNA sequencing, from sample preparation to bioinformatics and data analysis. If you have any questions, get in touch with team, who will be happy to help.

More 16S rRNA Sequencing Resources

• 16S sequencing sample report
• Video seminar: amplicon workflows
• Are you planning a microbiome study? Download our guide: How to plan and conduct a microbiome study: