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Fungal ITS Sequencing

Overview

CD Genomics' Fungal ITS Sequencing provides precise species-level identification using optimized primers and advanced sequencing technologies. We deliver high-resolution data for detailed fungal community analysis, including diversity and phylogenetics. Our service combines cutting-edge technology with expert support to offer comprehensive insights into fungal ecosystems, aiding in research and ecological studies.

Our Advantages:

High Resolution: Our sequencing approach delivers high-resolution data, allowing precise identification of fungal species and detailed characterization of fungal communities.
Comprehensive Analysis: We provide a thorough analysis of fungal diversity, including species-level identification and community composition, which is crucial for ecological and clinical research.
Advanced Technology: Utilizing cutting-edge sequencing platforms, such as Illumina, ensures high-quality data with minimal errors and high throughput.
Expert Support: Our experienced team of biologists and bioinformaticians offers expert guidance and support throughout the entire sequencing process, from sample preparation to data interpretation.

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What is an ITS Sequence

Internal Transcribed Spacer (ITS) sequences are critical components of ribosomal RNA (rRNA) genes in eukaryotes. These non-coding regions, specifically ITS1 and ITS2, are situated between the conserved coding regions of rRNA genes. In fungal genomes, ITS1 is located between the 18S and 5.8S rRNA genes, while ITS2 lies between the 5.8S and 28S rRNA genes. Notably, these regions exhibit considerable variability and are subject to fewer selective pressures compared to coding regions, making them particularly valuable for the study of fungal diversity and phylogeny. With lengths typically ranging from 500 to 750 base pairs, ITS sequences display significant sequence variability that mirrors evolutionary changes across fungal taxa. Consequently, ITS sequences serve as indispensable markers in fungal taxonomy, providing profound insights into the systematics and phylogenetic relationships among fungal species.

What is Fungal Sequencing

Fungal sequencing leverages advanced high-throughput sequencing technologies to analyze fungal DNA, including the ITS regions and the 28S rRNA gene. This process is integral for accurately identifying fungal species, understanding fungal community structures, and investigating their ecological roles within various environments.

Among the ITS regions, ITS1 and ITS2 are of particular importance. As non-coding regions, they experience less selective pressure and exhibit greater variability, providing the genetic diversity needed for detailed phylogenetic analyses. Recent studies suggest that ITS1 is often more effective than ITS2. ITS1 demonstrates higher efficiency, broader primer applicability, and a shorter amplification fragment with lower GC content compared to ITS2. Consequently, ITS1 may offer superior advantages for fungal sequencing, enhancing both the resolution and accuracy of fungal identification.

By utilizing NGS technologies, researchers can generate comprehensive profiles of fungal communities from environmental samples, clinical specimens, and other sources.

16S-ITS Association Study

Combining ITS and bacterial 16S rRNA sequencing, often referred to as the 16S-ITS association study, introduces a powerful framework for thoroughly analyzing microbial communities. 16S rRNA sequencing specializes in exploring bacterial populations. In contrast, ITS sequencing zeroes in on fungal communities. Merging these datasets, researchers achieve a panoramic view of the microbial ecosystem, revealing complex interactions between bacteria and fungi within a specific environment.

This integrative method significantly enhances our grasp of microbial diversity, ecosystem dynamics, and interspecies relationships. For instance, it can uncover how bacterial and fungal communities co-evolve, influencing each other's ecological roles. Such insights are critical for advancing our understanding of various contexts, be it environmental or health-related, providing a richer, more nuanced picture.

Applications

Species Evolution
Evolutionary Analysis
Species Abundance
Gene Discovery
Environmental and Ecological Research
Disease and Personalized Medicine

Service Specifications

Introduction to Our Fungal ITS Sequencing Service

At CD Genomics, our fungal ITS sequencing service utilizes advanced platforms like Illumina, PacBio, and Oxford Nanopore to deliver precise fungal identification and diversity analysis. Among these, the PacBio and Oxford Nanopore platforms are primarily used for Full-Length 16S/18S/ITS Sequencing. We ensure high-quality results through meticulous sample preparation and expert data analysis, providing detailed insights into fungal community structure and dynamics for applications in environmental and research settings.

Fungal ITS Sequencing Workflow

The Workflow of Fungal ITS Sequencing.

Technical Parameters

Sequencing Platform	Sequencing Strategy	Data Volume
HiSeq, MiSeq	PE300	Depending on the specific project requirements, not less than the contracted data volume

Bioinformatics Analysis

After DNA sequences are obtained, bioinformatics tools such as QIIME and mothur are commonly used for raw data processing, taxonomic assignment, diversity and richness analysis, comparative analysis, and evolutionary analysis. We are flexible to your needs.

Pipeline	Analysis Content
Raw data processing	Filtering and trimming of poor-quality sequences, read alignment
Taxonomic assignment	Operation Taxonomic unit (OTU) clustering and taxonomic assignment
Diversity and richness analysis	Rank abundance, Venn, alpha diversity analysis, beta diversity analysis, rarefaction curves, heatmap, etc.
Comparative analysis	Metastats, LEfSe, PCA analysis, PCoA analysis, etc.
Evolutionary analysis	(Un)weighted UniFrac analysis, phylogenetic trees
Custom analysis	CCA analysis, PICRUSt, network analysis, etc.

Note: The above content includes only a portion of the bioinformatics analysis. For more information or to customize the analysis, please contact us directly.

Sample Requirement

Sample Type	Quantity	Concentration	OD260/OD280
Tissue Sample	>1.5 g	-	-
Genomic DNA	≥200 ng	≥6 ng/μl	≥1.8
PCR Product	≥1 μg	≥10 ng/μl	-

Sampling kits: We provide a complete range of microbial sampling kits for clients, including microbial collection products, DNA/RNA isolation kits, and accessories for storage and mailing.

Note: If you wish to obtain more accurate and detailed information regarding sample requirements, please feel free to contact us directly.

Deliverables

Raw sequencing data (FASTQ)
Trimmed and stitched sequences (FASTA)
Quality-control dashboard
Statistic data
Your designated bioinformatics report.

Demo

Partial results of our fungal its sequencing service are shown below:

The Fungal ITS Sequencing Results Display Figure.

FAQs

Fungal ITS Sequencing FAQ

1. What is the difference between ITS1 and ITS2?
- ITS1 and ITS2, non-coding regions nestled within the ribosomal DNA of fungi, are located between the 18S and 28S rRNA genes. ITS1, positioned between the 18S rRNA and 5.8S rRNA genes, exhibits considerable sequence variation, making it invaluable for species identification and phylogenetic analysis. ITS2, situated between the 5.8S rRNA and 28S rRNA genes, also shows variability but typically maintains a more conserved secondary structure, offering stability for differentiating closely related species.
2. What types of samples can be used for fungal ITS sequencing?
- We accept an extensive variety of samples, including soil, water, air, and clinical specimens. To achieve optimal results, it is crucial that samples are handled according to our guidelines to maintain DNA integrity.
3. Can you provide customized analysis or additional data?
- Absolutely, we offer bespoke analysis services tailored to meet specific research requirements. Please reach out to us to discuss your particular needs.
4. How can I get started with your fungal ITS sequencing service?
- Start by contacting CD Genomics to discuss your project. Our team will guide you through the sample submission process and provide all the necessary information to ensure a successful analysis.

Case Study

Identification of the Fungal Community in Otomycosis by Internal Transcribed Spacer Sequencing
Impact factor: 5.640
Published: 15 March 2022

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Background

Otomycosis, a fungal ear infection common in humid, tropical regions, affects 10–20% of ear infections. It often involves Aspergillus species and Candida albicans. Traditional fungal identification methods have limitations, but ITS sequencing provides more accurate results. Topical antifungals are effective, even with ear membrane damage.

Materials & Methods

Sample preparation:

Otomycosis patients
Fungal mycelium-like discharge
RNA extraction

Method:

ITS sequencing
Illumina

Data Analysis:

Alpha- and Beta-diversity analyses
Linear discriminant analysis
Effect size analysis
Statistical analysis

Results

Principal Coordinates Analysis (PCoA) revealed distinct fungal community structures between pre-treatment and other groups, with the pre-treatment group being notably different from both control and post-treatment groups. Taxonomic analysis showed that Aspergillus was dominant in the pre-treatment group, while Malassezia was more prevalent in the control and post-treatment groups. Significant fungal taxa differences were observed between pre- and post-treatment samples, with Eurotiales and Aspergillus prevalent in pre-treatment, and Nectriaceae and Agaricales in post-treatment. Correlation analysis linked Aspergillus with symptoms like itching and hearing loss, whereas Malassezia showed negative correlations with these symptoms.

Figure 1. Principal coordinates analysis (PCoA) of fungal composition based on weighted UniFrac in three groups.

Figure 2. Phylum and genus level distribution of dominant fungi in the three groups. (Gu et al., 2022) Figure 2. Distribution of predominant fungi in three groups at the phylum and genus levels.

Figure 3. LEfSe analysis of fungal species. The bar graph shows the LDA scores calculated for characteristics at the OTU level.

Conclusions

In Nanjing otomycosis patients, Aspergillus was the main pathogen, while Malassezia was prevalent in healthy ears. Fungal diversity was lower in patients but increased after treatment. ITS sequencing effectively profiles fungal communities but has limitations in species-level identification; advanced sequencing methods are recommended for more accurate identification.

References

R. C. Summerbell, et. al. rRNA Gene Internal Transcribed Spacer 1 and 2 Sequences of Asexual, Anthropophilic Dermatophytes Related to Trichophyton rubrum. J Clin Microbiol. 1999, 37(12): 4005–4011.
A. J. Buehler, et. al. Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products. Journal of Dairy Science. 2017,100(11): 8814-8825.
Kõljalg U, et. al. UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi. New Phytol. 2005, 166(3):1063-8.
Gu X, Cheng X, Zhang J, et al. Identification of the fungal community in otomycosis by internal transcribed spacer sequencing. Frontiers in microbiology. 2022, 15;13:820423.