18S Amplicon Sequencing: Case Analysis in Marine, Soil, Sewage, and Pharmaceutical Fields

Introduction to 18S Amplicon Sequencing

18S Amplicon Sequencing is a high-throughput sequencing technology based on the 18S rRNA gene, playing a pivotal role in microbial ecology research. As an essential component of the small subunit of eukaryotic ribosomes, the 18S rRNA gene exhibits high conservation while also containing variable regions. The sequence variations in these variable regions provide crucial evidence for classifying and identifying different eukaryotic microorganisms. Compared to traditional microbial research methods, such as culture isolation and microscopic observation, 18S Amplicon Sequencing offers distinct advantages. It eliminates the need for microbial cultivation and can directly obtain genetic information from environmental samples, thereby providing a more comprehensive and accurate reflection of the true composition and diversity of microbial communities.

This article looks at this technology. It shows its wide use and great achievements in microbial research through real - world cases in different fields. These fields include marine phytoplankton, soil fungi, microbes in sewage treatment systems, and pharmaceutical R&D. By doing this, it shows the great value of 18S Amplicon Sequencing in understanding the roles of microbes and solving practical problems.

Case 1: 18S Amplicon Sequencing for Marine Phytoplankton Community Structure Study

In the study of marine ecosystems, phytoplankton, as primary producers, play a pivotal role in the overall marine ecological balance. With its high-throughput and high-sensitivity characteristics, 18S Amplicon Sequencing technology can directly obtain the genetic information of phytoplankton from marine water samples. This provides a powerful means for in-depth exploration of marine phytoplankton community structure, diversity, and their relationships with environmental factors, helping us better understand the functions and stability of marine ecosystems.

Title: "18S rRNA Analysis Reveals High Diversity of Phytoplankton with Emphasis on a Naked Dinoflagellate Gymnodinium sp. at the Han River (Korea)"

Journal: Diversity

Impact Factor: 3.031

Publication Date: February 10, 2021

DOI: 10.3390/d13020073

Sample Selection: The study selected phytoplankton samples from the Han River in Korea, with samples collected from monthly sampling in 2012 and 2019.

Research Technology: It adopted 18S Amplicon Sequencing technology.

Background: Phytoplankton play a crucial role in marine ecosystems, and their diversity is essential for understanding ecosystem health and functionality. Traditional microscopy methods have limitations in monitoring phytoplankton, while 18S rRNA amplicon sequencing technology offers a more precise and high-throughput alternative approach.

Objective: To reveal the seasonal diversity of phytoplankton in the Han River, particularly the distribution of Gymnodinium sp.

Research Approach and Results: The research team analyzed the seasonal dynamics of phytoplankton in the Han River through 18S rRNA amplicon sequencing. The results showed significant differences in phytoplankton diversity among different seasons, with the highest diversity in autumn. The study also found that Gymnodinium sp. dominated in autumn and was not detected in other seasons. In addition, through molecular cloning and qPCR techniques, the research team confirmed the presence of Gymnodinium sp. and found it in the autumn samples of both 2012 and 2019.

Impact: This study demonstrates the application potential of 18S rRNA amplicon sequencing technology in marine phytoplankton research, enabling more accurate monitoring of phytoplankton diversity and dynamic changes. This is of great significance for water quality management and the sustainable development of ecosystems.

Utilization of 18S Amplicon Sequencing for Research on Marine Phytoplankton Community Structure (Muhammad et al., 2021)

Case 2: 18S Amplicon Sequencing Applied to Soil Fungal Diversity Research

Soil is one of the ecosystems with the richest biodiversity on Earth, among which fungi, as an important microbial group, play a key role in soil nutrient cycling, plant growth promotion, and ecosystem stability. 18S Amplicon Sequencing technology can directly sequence and analyze the fungal 18S rRNA gene in soil samples, accurately identifying a large number of fungal species that are difficult to culture using traditional methods. This provides new perspectives and methods for in-depth research on soil fungal diversity and its ecological functions.

Title: "Profiling the eukaryotic soil microbiome with differential primers and an antifungal peptide nucleic acid probe (PNA): Implications for diversity assessment"

Journal: Applied Soil Ecology

Impact Factor: 4.8

Publication Date: August 2024

DOI: 10.1016/j.apsoil.2024.105464

Sample Selection: The study selected soil samples from farmland and forest areas.

Research Technology: It employed 18S Amplicon Sequencing technology, combined with two different primer pairs (TAReuk and EKeuk) and a new antifungal peptide nucleic acid probe (PNA).

Background: 18S rRNA gene amplicon analysis is an important tool for characterizing the diversity of soil eukaryotic microbial communities. However, different primer pairs and PNA probes have varying effects in detecting fungi and protists.

Objective: By using different primer pairs and PNA probes, the study aimed to investigate the diversity of soil eukaryotic microbial communities and evaluate the effect of the PNA probe in enhancing protist detection.

Research Approach and Results: Through in silico analysis and actual soil DNA experiments, the research team found that the TAReuk primer pair had high specificity for the protist SAR supergroup and Animalia, while the EKeuk primer pair had high specificity for Ascomycota and Basidiomycota. In silico analysis showed that the PNA probe could match the 18S rRNA genes of most Ascomycota (81.3%) and Basidiomycota (65.4%). However, in actual soil DNA experiments, the performance of the PNA probe was not as expected, especially showing differences in the detection of fungi and protists across different soil types. Nevertheless, the PNA probe could enhance the detection of specific protists (such as Conosa) in certain cases.

Impact: This study emphasizes the importance of selecting appropriate primer pairs and PNA probes in soil fungal diversity research. 18S rRNA amplicon sequencing technology has broad application prospects in soil fungal diversity research, but the specificity of primer pairs and PNA probes, as well as differences in soil types, need to be considered.

Case 3: 18S Amplicon Sequencing in the Study of Microbial Community in Sewage Treatment Systems

Sewage treatment plays a pivotal role in protecting water resources and preventing water pollution, with the microbial communities within sewage treatment systems serving as the core force for achieving sewage purification. Gaining an in-depth understanding of the structure and function of microbial communities in sewage treatment systems is of utmost importance for optimizing sewage treatment processes and enhancing treatment efficiency and quality. 18S Amplicon Sequencing technology, with its high-throughput and high-resolution characteristics, can rapidly and accurately identify various microorganisms in sewage treatment systems, providing strong support for studying the dynamic changes and functions of microbial communities during the sewage treatment process.

Title: "An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads"

Journal: Microbial Ecology

Impact Factor: 3.3

Publication Year: 2017

DOI: 10.1007/s00248-017-0996-9

Sample Selection: The study selected samples from a variety of organisms, including Cryptosporidium parvum, Toxoplasma gondii, Blastocystis hominis, Giardia intestinalis, rats, chickens, dogs, and horses. Additionally, samples were collected from wild birds captured in California, USA. Sewage samples were obtained from an aeration feed tank in the basement of a private apartment building in New York City.

Research Technology: The study employed 18S Amplicon Sequencing technology, combined with Sanger sequencing and Illumina MiSeq sequencing.

Background: 18S rRNA gene amplicon sequencing is an important tool for characterizing the diversity of microbial eukaryotic communities, especially in sewage treatment systems. However, different primer pairs and sequencing methods exhibit varying effectiveness in detecting eukaryotes.

Objective: To develop a reliable and optimized workflow for detecting and analyzing microbial eukaryotes in urban sewage samples, with a particular focus on zoonotic and trichomonad groups.

Research Approach and Results: The research team enhanced the efficiency and accuracy of detecting and analyzing microbial eukaryotes from urban sewage samples by optimizing experimental methods. They developed a sequencing workflow targeting the V4 and V9 hypervariable regions of the 18S rRNA gene, utilizing various experimental techniques, including bead-based PCR purification and size-based single-sample quantification, to generate high-quality sequencing libraries. By diluting and pooling individual libraries for sequencing, they achieved more uniform sequencing coverage across samples and reduced the amount of data lost during downstream processing. Furthermore, the research team discovered that adjusting the lower loading concentration based on the final concentration of the library pool could increase overall data output and decrease the need for PhiX control. They were able to generate read data of comparable or higher quality to a standard Illumina run using only 6% PhiX (V9 region, V2 kit). This strategy maximized the amount and quality of data generated, reduced the space used for sequencing PhiX, and increased coverage depth.

Impact: This study highlights the application potential of 18S rRNA amplicon sequencing technology in researching microbial communities within sewage treatment systems, enabling more accurate monitoring of microbial eukaryotic diversity and dynamic changes. This is of great significance for the management and optimization of sewage treatment systems.

Utilization of 18S Amplicon Sequencing for Research on Microbial Communities in Wastewater Treatment Systems (Maritz et al., 2017)

Case 4: 18S Amplicon Sequencing Instances within Pharmaceutical Research

In recent years, with the rapid development of molecular biology technologies, 18S Amplicon Sequencing has become a crucial tool for studying microbial diversity, ecological functions, and disease-related microbial communities. In the field of pharmaceutical research, 18S Amplicon Sequencing is not only used to analyze microbial contamination in drug production environments but is also widely applied in research on drug-microbiome interactions. This technology can reveal the composition and function of microbial communities, providing an important molecular foundation for drug development, disease diagnosis, and precision medicine.

Title: N6-methyladenosine (m⁶A) in 18S rRNA promotes fatty acid metabolism and oncogenic transformation

Journal: Nature Metabolism

Impact Factor: 18.9

Publication Date: August 23, 2022

DOI: 10.1038/s42255-022-00622-9

Sample Selection: The study selected liver cancer cell lines (HepG2, Huh7), normal liver cell lines (THLE-2), and tumor and non-tumor tissue samples from liver cancer patients.

Research Techniques: Techniques such as 18S rRNA m⁶A modification analysis, Western blot, qPCR, lipid metabolism analysis, and translation efficiency analysis were employed.

Background: N6-methyladenosine (m⁶A) is one of the most abundant modifications in eukaryotic rRNA, yet its role in translation regulation remains unclear. METTL5 is the key enzyme that catalyzes the m⁶A modification of 18S rRNA, but its role in cancer has not been thoroughly investigated.

Objective: To investigate the function of METTL5-mediated 18S rRNA m⁶A modification in liver cancer, particularly its impact on fatty acid metabolism and tumor transformation.

Research Approach and Results: The research team found that METTL5 was highly expressed in liver cancer tissues and was associated with poor prognosis. METTL5 promoted the assembly of 80S ribosomes and global mRNA translation by catalyzing the m⁶A modification of 18S rRNA. Additionally, knockdown of METTL5 significantly reduced the levels of polyunsaturated fatty acids (PUFAs) in liver cancer cells and inhibited fatty acid β-oxidation. These results indicate that METTL5-mediated 18S rRNA m⁶A modification promotes tumor transformation in liver cancer by regulating fatty acid metabolism.

Impact: This study reveals the important role of METTL5 in liver cancer, particularly its mechanism of influencing fatty acid metabolism by regulating 18S rRNA m⁶A modification. These findings provide new potential therapeutic targets for the treatment of liver cancer.

Conclusion

In summary, through a detailed analysis of five case studies across diverse fields, we've comprehensively demonstrated the extensive applications and immense potential of 18S Amplicon Sequencing technology in microbial research. Whether in conventional environments like oceans, soils, and sewage treatment systems, or specialized areas such as pharmaceutical R&D and extreme habitats, this technology consistently provides researchers with rich insights into microbial community composition. It aids in uncovering the relationships between microbes and their environments, elucidating microbial functions, and clarifying their roles in ecosystems.

With its advantages of high throughput, sensitivity, and accuracy, 18S Amplicon Sequencing has become an indispensable tool in microbial ecology studies. As the technology continues to advance—evidenced by longer read lengths, further reduced sequencing costs, and optimized bioinformatics analysis methods—it will play an even more pivotal role in future microbial research. This progress will not only deepen our understanding of the microbial world but also provide robust technical support and theoretical foundations for addressing environmental challenges, developing novel drugs, and ensuring ecological security.

For these reasons, we have every confidence that 18S Amplicon Sequencing technology will continue driving advancements in microbial research, making significant contributions to the broader life sciences field.

References:

1. Muhammad BL, Kim T, et al. "18S rRNA Analysis Reveals High Diversity of Phytoplankton with Emphasis on a Naked Dinoflagellate Gymnodinium sp. at the Han River (Korea)." Diversity. 2021;13(2):73. https://doi.org/10.3390/d13020073
2. Wang H, Dumack K, et al."Profiling the eukaryotic soil microbiome with differential primers and an antifungal peptide nucleic acid probe (PNA): Implications for diversity assessment." Applied Soil Ecology. 2024;200:105464. https://doi.org/10.1016/j.apsoil.2024.105464
3. Maritz JM, Rogers KH, et al. "An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads." Microb Ecol. 2017;74(4):923 - 936. https://doi.org/10.1007/s00248-017-0996-9
4. Peng H, Chen B, et al."N6-methyladenosine (m6A) in 18S rRNA promotes fatty acid metabolism and oncogenic transformation." Nat Metab. 2022;4(8):1041 - 1054. https://doi.org/10.1038/s42255-022-00622-9