Full-length 18S / ITS Amplicon Sequencing Data Analysis

What is 18S /ITS Amplicon Sequencing Analysis?

16S/18S/Internal Transcribed Spacer (ITS) amplicon sequencing is widely used in microbial research because of its low cost, short cycle time, and low sample quality requirements. Among them, 18S and ITS sequencing is commonly used to identify fungi and other eukaryotes in environmental samples. Both methods use PCR to amplify DNA with specific primers, and the results are obtained after sequence processing, sequence analysis, and comparison. ITS sequencing is more focused on studying the intraspecific genetic diversity of fungi due to greater ITS variability, while 18S rRNA focuses on phylogenetic taxonomic studies of fungi. In recent years, scientists are developing full-length 18S/ITS amplicon sequencing methods based on PacBio and Oxford Nanopore sequencing technologies to identify a wider range of species and distinguish strains within species.

Fig. 1. Taxonomic classification of eukaryotic microbes using 18S amplicon sequencing. (Popovic A, et al, 2018)

Application of Full-length 18S / ITS Amplicon Sequencing Analysis

Our full-length 18S/ITS amplicon sequencing analysis can be used for but not limited to the following research:

Environmental microbiology: our full-length 18S/ITS amplicon sequencing analysis can help you characterize microbial communities in the environment and reveal their role in nutrient cycling, bioremediation, and overall ecosystem function.

Human microbiome research: our full-length 18S/ITS amplicon sequencing analysis can characterize microbial communities in different body parts, such as the gut, mouth, skin and reproductive tract. This analysis helps reveal the complex relationships between the human microbiome and various health conditions.

Agriculture and food safety: our full-length 18S/ITS amplicon sequencing analysis can be used to study microbial communities associated with crop health, soil fertility and plant-microbe interactions. It also helps to detect and monitor foodborne pathogens and spoilage microbes, ensuring food safety and quality throughout the production and distribution chain.

Biotechnology and pharmaceutical research: our full-length 18S/ITS amplicon sequencing analysis allows monitoring and characterizing these microbial communities, helping you to identify potential contaminants, optimize production conditions, and develop effective microbial control strategies.

Full-length 18S/ITS Sequencing Process

Fig 2 The Process of Full-length 18S/ITS Sequencing

Sample Submission Guidelines of Sequencing

CD Genomics Data Analysis Pipeline:

CD Genomics is a leading company in genomics and sequencing services, providing comprehensive and advanced bioinformatics analysis of full-length 18S/ITS amplicon sequencing data. Utilizing cutting-edge technology and expertise in the field, CD Genomics assists researchers and scientists in unraveling the complexity of microbial communities and their functions.

(1) Data pre-processing

Quality control and filtering of raw sequencing data obtained by sequencing full-length 18S/ITS amplicons to remove low-quality reads, adapter sequences and artifacts. CD Genomics employs stringent quality control measures to ensure reliable downstream analysis.

(2) Sequence alignment and clustering

CD Genomics performs sequence alignment using specialized algorithms and databases to map sequencing reads to reference databases, such as SILVA or UNITE for the 18S and ITS regions, respectively.

(3) Taxonomic analysis and diversity analysis

CD Genomics uses advanced bioinformatics tools to assign taxonomic identities to OTUs and generate taxonomic analysis histograms and abundance heat maps.

(4) Functional analysis:

CD Genomics can predict the abundance of functional pathways based on 16S copy number.

(5) Statistical analysis and visualization

CD Genomics uses various statistical methods and visualization techniques to reveal significant differences and relationships in the data. Multivariate statistical analyses are used to identify changes in microbial communities under different conditions or treatments. In addition, visualization methods such as principal component analysis (PCA) and ternary plots facilitate visualization and interpretation of complex microbial data sets.

Full-length 18S / ITS Amplicon Sequencing Analysis Content:

What Are the Advantages of Our Services?

Stringent Quality Control

Our protocols are meticulously designed to ensure high-quality RNA extraction and effective ribosomal RNA removal, which are crucial for obtaining accurate sequencing results.

Reliable Data

We maintain data integrity by implementing rigorous quality assessment steps, including adapter trimming and the removal of low-quality sequences.

State-of-the-Art Sequencing Platforms

By utilizing cutting-edge sequencing technology, we achieve high-throughput and precise 18S/ITS amplicon sequencing.

Innovative Techniques

Our advanced methodologies significantly enhance the detection of target RNA molecules, thereby improving the sensitivity and specificity of our sequencing processes.

Sophisticated Bioinformatics Tools

Our comprehensive data analysis pipeline includes quality control, sequence alignment, and functional annotation, ensuring detailed insights into microbial communities.

Functional and Pathway Analysis

We conduct in-depth analysis of differentially expressed genes and their enrichment in biological pathways, elucidating the functional roles of microbial species.

Example Data Analysis Report

To demonstrate the quality and detail of a CD Genomics report for Full-length 18S Amplicon data analysis, we have a sample report available. Contact us to request our Full-length 18S Amplicon data report. You can also refer to a client-published article, " Blocking IL-17A prevents oxycodone-induced depression-like effects and elevation of IL-6 levels in the ventral tegmental area and reduces oxycodone-derived physical dependence in rats." which includes some of the data we provided.

How It Works

CD Genomics is a high-tech company specializing in multiomic data analysis. We provide services such as project design, data analysis, and database construction. With a focus on developing breakthrough products and services, we are a pioneer in the biotechnology industry, serving researchers and partners worldwide.

CD Genomics provides valuable support and insight in areas such as these applications with its extensive experience and expertise in bioinformatics analysis of full-length 18S / ITS amplicon sequencing. We can help you gain a deeper understanding of the diversity, composition and function of your microbial community. From species annotation and diversity analysis to functional prediction and enterotype analysis, CD Genomics offers a comprehensive solution for unlocking the mysteries of the microbial world. If you are interested in our services, please contact us for more detailed information.

Reference

1. Popovic A, Parkinson J. Characterization of eukaryotic microbiome using 18S amplicon sequencing[J]. Microbiome analysis: methods and protocols, 2018: 29-48.

What Does Analysis of Full-length 18S / ITS Amplicon Sequenceing Show?

Data Processing and Statistics

Table 1 Data statistics of the quality control (Top 10).

Fig 3 Effective tags length distribution of sample ID C1_1_M.

Feature table construction

Fig 4 The frequency of each sample

Species Annotation and Taxonomic Analysis

Taxonomy Distribution Histogram of All Samples

Fig 5 The taxonomy distribution of all samples in the Phylum classification level.

Species Abundance Heatmap

Fig 6 Species abundance Heatmap. Phylum.

Alpha Diversity Analysis

Statistical Data of Alpha Diversity

Table 2. Statistics of Alpha diversity indices (Top 10).

Rarefaction Curve

Fig 7 Rarefaction curve of the sequenced reads for all samples (The above figure) and The depth of the sequencing samples (The below figure).

Beta Diversity Analysis

Boxplot Analysis

Fig 8 Boxplot analysis based on Bray Curtis.

PCoA Analysis

Fig 9 PCoA analysis based on Bray Curtis.

UPGMA Analysis

Fig 10 UPGMA clustering tree based on unweighted unifrac. The different colors represent different groups.

Significant Difference Analysis

ANCOM Analysis

Table 3. ANCOM analysis partial result in phylum-level in GROUP.

Fig 11 Mean proportion of treated and control group.

Title: PacBio next-generation sequencing uncovers Apicomplexa diversity in different habitats

Publication: Scientific Reports

Main Methods: full-length 18S rRNA amplicon sequencing

Abstract:

The phylum Apicomplexa includes intracellular protozoan parasites causing diseases like toxoplasmosis and cryptosporidiosis. This study used next-generation 18S rRNA amplicon sequencing with PacBio technology to explore Apicomplexa diversity in riverine environments. PCA, PCoA, PERMANOVA, ANOSIM, Cluster analysis, and Venn diagrams indicated habitat heterogeneity. Dominant genera in inlet samples were Gregarina, Cryptosporidium, and Leidyana, while outlet samples were dominated by Babesia, Cryptosporidium, and Theileria. Surface water had 16% and 8.33% relative abundance of Toxoplasma and Cryptosporidium, respectively. This is the first comprehensive study of these parasites in Egypt, aiding water quality standards.

Research Results:

The study identified 52,214 microeukaryotic OTUs from 136,127 high-quality reads. PCA showed clustering of physicochemical parameters by habitat, notably between inlet and surface water samples. Lower-quality outlet samples resembled inlet samples, while higher-quality outlet samples were similar to surface water samples. Significant differences in environmental parameters were confirmed by PERMANOVA and ANOSIM. The difference between inlet and surface water parameters was most pronounced compared to inlet vs. outlet and outlet vs. surface water.

Fig 12 Principal component analysis (PCA) plot for spatial variation of physicochemical parameters between different habitats (principal components (PC1 and PC2) explained 69.6% of the total variation).

Table 4. PERMANOVA and ANOSIM test the significance of the differences in physicochemical parameters between the habitats.

The PCoA and Cluster Analysis revealed significant differences in the Apicomplexa community across habitats, with inlet samples distinct from outlet and surface water samples. Apicomplexan species were less than 0.01% of microeukaryotic communities. Comparable patterns were observed in studies from China and the USA. The Venn diagram showed no shared apicomplexan species across habitats, with 89% unique species in inlet samples, and fewer unique species in outlet (4) and surface water samples (7). The study suggests that physicochemical parameters influence apicomplexan community distribution, necessitating further research on specific environmental factors.

Fig 13 Principal coordinate analysis (PCoA) based on Bray Curtis similarity index showing the β-diversity patterns of the Apicomplexa community

Fig 14 Cluster analysis of Apicomplexa OTUs in different environments

Fig 15 Venn shape shows the unique and shared Apicomplexa OTUs in different habitats.

The heatmap revealed more Apicomplexan genera in inlet samples compared to outlet and surface water samples, indicating effective parasite removal during wastewater treatment. This decrease in parasite abundance, from raw sewage to Nile River water, aligns with Freudenthal et al.'s findings on the efficacy of wastewater treatment technology in reducing health risks. Effective pathogen removal is crucial for safe wastewater reuse. Network analysis showed associations between parasitic protists (e.g., Dientamoeba, Entamoeba, Giardia) and potential predators (ciliates, rotifers), suggesting predation as a factor in parasite reduction.

Fig 16 Heatmap showing the distribution of Apicomplexa taxa in different environments.

The study found that the dominant apicomplexan parasites in inlet samples were Gregarina (38.54%), Cryptosporidium (32.29%), and Leidyana (11.90%), while outlet samples were dominated by Babesia (33.33%), Cryptosporidium (25%), and Theileria (16.67%). Apicomplexa were the most abundant protozoa in both inlet (57.2%) and outlet (46.9%) samples, similar to findings in New Zealand wastewater. The relative abundance of Cryptosporidium decreased from inlet to outlet, consistent with previous research showing wastewater as a hotspot for parasites.

Fig 17 Taxonomic composition of the Apicomplexa community. Others refer to unclassified and less contributed taxa.

Conclusions:

Utilizing next-generation 18S rRNA amplicon PacBio sequencing, this study investigated Apicomplexa in Egypt's aquatic habitats, detecting a wide range of parasites. Findings revealed the presence of Cryptosporidium in treated sewage and Nile River samples, indicating potential health risks due to insufficient wastewater treatment. The study suggests the need for national legislation on drinking water and wastewater reuse standards based on WHO guidelines. Further research is needed to explore microeukaryotic parasite diversity in other environments, such as drinking water and swimming pools, using advanced sequencing technologies.

1. What is 18S rRNA sequencing for?

The genes encoding 18S rRNA are known as 18S rRNA genes. These sequences are extensively utilized in molecular analyses to reconstruct the evolutionary history of organisms, particularly in vertebrates. Due to their slow rate of evolution, 18S rRNA genes are ideal for studying ancient divergences.

2. What is the size of 18S rRNA amplicon?

The 18S rRNA gene in eukaryotic microbes, ranging in length from 1500 to 2000 base pairs, contains both conserved and variable regions.

3. What is the difference between ITS and 18S sequencing?

ITS amplicon sequencing is more suitable for studying fungal diversity, offering high accuracy in species identification. In contrast, 18S amplicon sequencing targets the diversity of eukaryotic microbes, providing a broader range of species annotations but with relatively lower accuracy.

4. How do 16S rRNA and 18S rRNA differ from each other?

Although both 16S and 18S rRNA are crucial for ribosome function and protein synthesis, they primarily differ in the organisms they are found in and their uses in molecular biology. Specifically, 16S rRNA is used for identifying and classifying prokaryotic organisms, while 18S rRNA serves the same purpose for eukaryotic organisms.

5. How were the Full-length 18S/ITS Amplicon sequencing data analyzed?

The process begins with merging raw data, performing quality checks, filtering, and removing chimeras to create ASV clusters. This is followed by alpha diversity analysis (including rarefaction, Chao1, and Shannon curves), species annotation (using KRONA and phylogenetic trees), and beta diversity analysis (such as UniFrac distances and PCA). Diversity statistics are then calculated (using NMDS and DCA). Advanced analyses include Metastats, LEfSe, and RDA/CCA.