Amplicon sequencing entails the amplification of specific DNA fragments through PCR technology, followed by the sequencing of these amplified products. This methodology is primarily employed to address the question of "which microorganisms are present." By amplifying and sequencing high-variable regions of 16S, 18S, ITS, and functional genes within a sample, microbial composition can be identified at the genus/species level. It is imperative to note that the target of amplicon sequencing is the genetic material in the form of DNA.
Bacterial 16S rDNA
The ribosomes of prokaryotes consist of 5S rRNA, 23S rRNA, and 16S rRNA. Among these, the gene encoding 16S rRNA possesses distinct characteristics: ubiquity in prokaryotes, moderate size (1540 nt), high copy numbers, ease of template acquisition, and inclusion of both highly conserved and variable regions. These features render the 16S rRNA gene particularly well-suited for bacterial diversity analysis.
The sequence of 16S rDNA is characterized by the presence of nine conserved regions juxtaposed with nine variable regions. The preference for a specific sequencing region is largely influenced by the inherent attributes of the sample under investigation.
More precisely, for bacterial diversity assessment, the V3-V4 region of the 16S rDNA sequence is frequently utilized. This particular region is particularly effective when analyzing intestinal specimens due to its high level of suitability.
In contrast, the V5-V7 region proves beneficial when examining samples likely to contain plant host contaminants, specifically, endophytic bacteria, due to its unique attributes.
The V4-V5 region, however, is distinctly suitable when dealing with environmental specimens such as soil and sediment. This suitability arises from the region's inherent characteristics that enable elucidation of environmental microbial diversity present in these samples.
Therefore, these regions are selected based on their unique attributes that allow for effective bacterial diversity assessment, thereby contributing to the ever-expanding field of microbiome research. The process of choosing an appropriate sequencing region is rooted in its applicability and potential to deliver accurate, insightful data instrumental in advancing our understanding of bacterial diversity.
16S Sequencing Workflow
The sequencing workflow of amplicon sequencing technologies generally exhibits fundamental consistency. Taking the 16S amplicon sequencing process as an illustrative example, we can delineate its procedural steps in detail. Initially, total DNA is extracted from the sample. Subsequently, primers designed based on conserved regions are employed for PCR amplification. Following this, the amplified products undergo purification, quantification, and normalization, culminating in the formation of a sequencing library. Subsequent steps involve quality control, sequencing, and data analysis. The 16S amplicon sequencing workflow is illustrated in the figure below.
Eukaryotic 18S rDNA
Within eukaryotes, the V4 region database of 18S rDNA encapsulates the most comprehensive information and demonstrates excellent classification efficacy. Consequently, it is widely acknowledged as the optimal choice for the annotation and analysis of the 18S rRNA gene, particularly for taxa such as algae and protozoa within the eukaryotic domain.
Fungal ITS
The Internal Transcribed Spacer (ITS) sequence, situated between the fungal 18S, 5.8S, and 28S rRNA genes, comprises two distinct segments: ITS1 (350 bp) and ITS2 (400 bp).ITS1 is conventionally favored, whereas ITS2 finds applicability in the identification of fungi within samples such as those derived from fermentation processes and environmental matrices like soil.
Functional Gene Amplicon Sequencing
Functional microorganisms refer to microbes endowed with specific biological functions. While these microorganisms may exhibit considerable taxonomic diversity, they share analogous genes that enable them to perform similar functions. Consequently, genes that facilitate the expression of specific functions in functional bacteria, such as nxrA, nirS/nirK, amoA, dsrB, nifH, and others, are collectively referred to as functional genes. The process of primer design, amplification, and sequencing targeting these functional genes is termed Functional Gene Amplicon Sequencing.
| Functional Microorganisms |
Functional Genes |
| Nitrogen-fixing microorganisms |
nifH |
| Carbon-fixing microorganisms |
cbbL and cbbM |
| Nitrite-oxidizing bacteria (NOB) |
nxrA |
| Ammonia-oxidizing bacteria (AOB) |
amoA |
| Sulfate-reducing bacteria (SRB) |
dsrB |
| Methane-oxidizing bacteria |
pmoA |
| ... |
... |
