Amplicon sequencing has been an integral methodology for microbiologists and microbiome researchers for decades; enabling the generation of whole community analysis of bacteria or fungi in any complex sample.
By amplifying conserved variable regions of either the 16S gene or ITS gene, microbial communities can be differentiated and measured with high sensitivity at relatively low cost.
In doing so, this approach has enabled the field of microbiology to understand the layers of the microbiome for decades, leading to an even greater understanding of the microbial environment and its role in various functions, such as human health.
In this blog, we will cover what this popular sequencing method is, and the key benefits of Amplicon sequencing that can power your research.
What is Amplicon Sequencing?
Amplicon sequencing is the selection and isolation of genetic region(s) of interest before sequencing to illustrate the genetic variation between the samples within the target region.
What this essentially means is that targeted sequencing captures the genetic variation of microbial communities within a known genetic region which can then be compared between samples.
For most common applications, the 16S gene is isolated and amplified for sequencing to differentiate bacteria, while the ITS gene is isolated and amplified for sequencing to differentiate fungi.
Applications and Uses of Amplicon Sequencing
With amplicon sequencing, a wide range of applications for microbiome investigation become possible. Amplicon sequencing has been used in various microbiome research initiatives, including stool, skin, blood, and environmental applications.
By amplifying conserved variable gene regions that enable microbial differentiation, researchers are able to better investigate the microbial compositions of their samples of interest, both in regards to diversity as well as abundance of organisms.
Additionally, amplicon sequencing has also shown the possibility for having utility into infectious disease testing.
Whereas blood culture has the possibility of a negative result, amplicon sequencing of the 16S gene has the possibility to increase the sensitivity of detection to possible infectious agents in cardiovascular infections, as shown in this paper.
Amplicon sequencing has been a staple in microbiome research for decades. In its approach, microbial signatures of bacteria and/or fungi have been amplified for detection and characterization in various difficult sample types, such as blood, skin, environmental samples, without the need for significantly increased sequencing data for analysis.
As a result, this decrease in cost of sequencing enables researchers to consider increased sample numbers in their studies, allowing increased effect size for more significant determinations in the role of microbial communities.
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5 Key Benefits of Amplicon Sequencing
Benefit 1: Specificity
With amplicon sequencing, specific gene regions can be targeted for targeted sequencing and differentiation.
For example, variable regions of the 16S gene can be targeted in order to identify different organisms of interest, such as V1 region for Staphylococcus, V2 for Mycobacterium, V3V4 for various soil based organisms, etc.
With this information at hand, different amplicon primers can be used for research specific applications, allowing microbiome research to be more focused on organisms of interest, and less about insignificant organisms.
Benefit 2: Cost-effectiveness
With amplicon sequencing, one of the benefits is the ability to generate sequenced data only of genomic content of interest.
This is done during the library preparation of DNA samples, in which probes of the gene of interest (16S, ITS, etc) amplify these genes, with clean-up resulting in only genomic content of these specific genes.
As a result, when samples are sequenced, only the genes of interest will produce sequenced data for analysis. Because of this, all samples can be normalized for sequencing depth (typically 50k PE reads), which normally generates more than enough data for sufficient analysis of the genetic diversity within the sample across this specific gene region.
This amount of reads is significantly decreased compared to the amount of reads needed for metagenomic analysis, for example, as metagenomic analysis has to also compete with non-microbial reads (in addition to the microbial reads present within the sample).
With amplicon sequencing, any genetic content that is not within the targeted gene region will not be represented in the sequencing data, thus significantly cost-optimizing sequencing.
Benefit 3: Simplicity and speed
With amplicon sequencing, many aspects of next-generation sequencing and analysis are streamlined for processing, thus increasing the likelihood of success for project success. One of these aspects deals with DNA necessary for library preparation.
Since amplicon sequencing implements a PCR heavy approach (in an attempt to amplify the target gene sequence), this significantly decreases the amount of DNA needed to perform sequencing.
This allows for more difficult sample types with low DNA yields, such as skin swabs, to be successful for next-generation sequencing and analysis.
Benefit 4: High sensitivity
Another aspect of amplicon sequencing that is important to consider is the increased sensitivity to organisms within samples. In part due to the PCR amplification of the gene regions of interest, amplicon sequencing discards all “noise” of non-target genes within the sample, thus increasing the signal of microbial targets within the sample.
Benefit 5: Versatility
Amplicon sequencing provides the ability to target various organisms of interest through the selection of genes of interest (ITS for Fungal, 16S for Bacterial), as well as specific regions of these genes for better classification of organisms of interest (1 region for Staphylococcus, V2 for Mycobacterium, V3V4 for various soil based organisms, etc.).
With various research applications being explored with the microbiome, such as Skin microbiome (V1V3), and Stool microbiome (V3V4), amplicon sequencing has the ability to target genes as well as gene regions relevant for both the sample type as well as research application being investigated by your research.
Amplicon Sequencing Challenges
With amplicon sequencing, the main challenge that impacts its utility involves the accuracy of speciation, as well as abundance characterization.
Regarding speciation, the ability to differentiate organisms based on mutations within a single gene is sometimes not possible, thus resulting in the inability to differentiate down to species level (and sometimes speciation via amplicon sequencing is inaccurate).
As for abundance, amplicon sequencing is PCR dependent, thus the accuracy with regards to relative abundance of organisms within samples has the possibility of being skewed as a result of unforeseen amplification biases.
To mitigate these challenges, alternatives include full-length amplicon sequencing (in which all variable regions of a targeted gene region are sequenced – such as 16S), as well as metagenomic sequencing (which includes the sequencing of the total community DNA for analysis).
Closing Thoughts
In closing, amplicon sequencing is a cost-effective option available to microbiome researchers interested in investigating the microbial compositions of their samples.
Additionally, for samples typically plagued by issues such as high non-microbial DNA content and low DNA yield, amplicon sequencing offers an approach that enables researchers to generate genomic data for microbiome analysis and differentiation at a cost-effective rate.
Thus, if you are interested in generating data for microbiome research in a cost-effective manner, or in difficult to test samples, reach out to a CosmosID representative to kick-off a discussion around how we can provide a solution to fit your needs.
Unlock the Power of the Microbiome With CosmosID…
At CosmosID, our state-of-the-art microbiome sequencing platform and sequencing services are second-to-none.
Our Amplicon Sequencing Services not only provide you with the ability to identify and differentiate microbial communities, but also characterize changes in diversity over time through longitudinal studies.
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Amplicon Sequencing FAQs
What is the role of Polymerase Chain Reaction (PCR) in amplicon sequencing methods?
PCR is crucial to amplicon sequencing as it amplifies the specific genomic regions of interest. This enhancement of the target gene sequences allows for successful sequencing even in cases of low DNA yield.
How does amplicon sequencing compare to whole genome sequencing?
While whole genome sequencing provides a comprehensive view of all the genetic variations within a sample, amplicon sequencing focuses on sequencing specific genomic regions (like the 16s rrna gene). This targeted approach makes amplicon sequencing more cost-effective than whole genome sequencing.
Amplicon sequencing allows researchers to analyze genetic variations within specific genomic regions. By focusing on these regions, researchers can identify single nucleotide polymorphisms and understand the genetic diversity within a sample.
What are the most common amplicon sequencing methods?
A common amplicon sequencing method refers to the widely used practice of focusing on a specific genomic region such as the 16s rRNA gene. This method is commonly used in microbiome research to identify bacterial diversity and analyze genetic variation within a sample.
Next-Generation Sequencing (NGS) amplicon sequencing is a method that uses modern sequencing technology to generate high-throughput data of specific genomic regions. This method allows for rapid, cost-effective data analysis of genetic variations within a sample.
How are oligonucleotide probes used in targeted next-generation sequencing?
Oligonucleotide probes are used in targeted next-generation sequencing to isolate and amplify the specific genomic regions of interest. These probes bind to the complementary DNA sequences, allowing for sequencing of these regions.
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