In the realm of biological sciences, accurate species identification is paramount. Whether studying biodiversity, diagnosing plant diseases, ensuring food safety, or identifying human pathogens, precise species identification underpins effective research, analysis, and intervention. One powerful molecular method that has revolutionized species identification, particularly for fungi, is ITS sequencing.
Fig 1. ITS Sequencing for species identification of microbial communities (Dhiraj K., 2017)
What is ITS Sequencing
ITS sequencing is a molecular technique that utilizes the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA) as a genetic marker for species identification. The ITS region is a segment of non-functional DNA situated between the small subunit (SSU) and large subunit (LSU) rRNA genes. The ITS region is characterized by high sequence variability among different species, even those closely related, making it an ideal marker for species discrimination.
The Process of ITS Sequencing
The process of ITS sequencing involves several key steps:
- DNA Extraction: The first step involves extracting DNA from the sample of interest. This sample could be a fungal culture, tissue from a plant or animal, or an environmental sample like soil or water.
- PCR Amplification: The extracted DNA is then subjected to Polymerase Chain Reaction (PCR) to amplify the ITS region. Specific primers are used to target conserved regions flanking the ITS, ensuring selective amplification of the desired region.
- Sequencing: The amplified ITS region is sequenced using Sanger sequencing or next-generation sequencing technologies. This generates a unique DNA sequence for the species in question.
- Database Comparison: The obtained sequence is compared against extensive databases, such as GenBank or specialized fungal databases like UNITE. A match between the query sequence and a database entry leads to species identification.
Technical Principles of ITS Sequencing
- High Variability: The ITS region exhibits significant sequence variation across different species. This interspecific variation is crucial for distinguishing between even closely related species.
- Universal Presence: The ITS region is present in all eukaryotes, making ITS sequencing a versatile technique applicable to a broad range of organisms.
- Ease of Amplification: The relatively short length of the ITS region facilitates easy amplification using PCR, even from samples with degraded DNA.
Technical Features of ITS Sequencing
ITS sequencing boasts several technical features that make it a preferred method for species identification:
- Accuracy: ITS sequencing offers a high degree of accuracy in species identification.
- Sensitivity: It can successfully identify species even from small or degraded samples.
- Versatility: The technique is applicable to a wide range of fungi and other eukaryotic organisms.
- Rapid Turnaround Time: Results can be obtained relatively quickly, facilitating timely decision-making.
Technical Classifications
- ITS1 Sequencing: Focuses on sequencing the ITS1 region, located between the 18S and 5.8S rRNA genes.
- ITS2 Sequencing: Involves sequencing the ITS2 region, situated between the 5.8S and 28S rRNA genes.
The choice between ITS1 and ITS2 sequencing often depends on the specific group of organisms being studied and the availability of reliable reference databases.
Applications
- Fungal Identification: ITS sequencing is a cornerstone of fungal identification, playing a crucial role in mycology, plant pathology, food safety, and medical mycology.
- Environmental Monitoring: Analyzing fungal communities in soil, water, and air provides valuable insights into ecosystem health and biodiversity.
- Evolutionary Studies: ITS sequences contribute to understanding the evolutionary relationships between different species.
- Plant Identification: While less common than for fungi, ITS sequencing can also be used for plant species identification, especially in cases where morphological identification is challenging.
Environmental Benefits
- Biomonitoring: Assessing fungal diversity in environmental samples helps monitor ecosystem health and detect potential threats.
- Disease Diagnostics: Rapid identification of plant pathogens facilitates timely intervention, minimizing the use of chemical treatments and their environmental impact.
- Bioremediation: Identifying fungi with bioremediation potential aids in developing sustainable solutions for environmental pollution.
Our Services
At our bioenvironmental company, we offer a comprehensive range of ITS sequencing services designed to provide accurate species identification and detailed insights into microbial communities. Here's an overview of the services we provide:
- High-Quality Sequencing: We deliver sequencing results from extraction in as little as 2 weeks, ensuring you receive your data promptly.
- Species-Level Resolution: Utilizing a curated 16S rRNA database, our 16S amplicon sequencing service achieves taxonomic identification down to the species level, providing unparalleled precision in species identification.
- Abundance Quantification: Our service goes beyond relative abundance, offering absolute abundance quantification to reveal more about microbial community dynamics, which is crucial for understanding treatment or therapeutic effects.
- Comprehensive Bioinformatics Analysis: Each project includes a detailed report with bioinformatics analysis and raw sequencing data. We provide compositional bar plots, taxonomy heatmaps, alpha-diversity and beta diversity analyses, and LEfSe biomarker discovery.
- Library Preparation and Quality Control: We employ 96 Magbead DNA and DNA Miniprep for DNA extraction, and Quick-16S NGS library prep for library preparation. We also include positive controls for DNA extraction and library preparation, ensuring stringent quality control.
- Sequencing Analysis: We use Amplicon Sequence Variant (ASV) based analysis with DADA2, providing higher accuracy and better resolution of data.
- Data Visualization and Statistical Analysis: We offer a range of data visualization tools, including absolute abundance boxplots, taxa abundance barplots, heatmaps, and alpha and beta diversity plots. Statistical analysis with LEfSe uncovers taxa/traits that are differentially distributed among sample groups.
Distinctive Service Features
- Industry-Leading Standards: We maintain high sequencing standards, with up to ≥80% of bases having a ≥Q30 quality score, depending on the chosen sequencing strategy.
- Unique Sequencing Technology: Our services leverage unique sequencing technology, which offers lower amplification error rates and much lower duplication rates compared to other platforms .
- Experienced Laboratory Professionals: We are backed by a team of highly experienced laboratory professionals who follow strict quality procedures to ensure the integrity of your results .
- Educational Resources and Support: We provide access to educational resources, including webinars and sample preparation guides, to support our clients throughout their projects.
Contact Us
By choosing our ITS sequencing services, you benefit from a combination of advanced technology, expert analysis, and a client-focused approach that delivers reliable and actionable insights into your microbial samples, please contact us and confirm with our team for more information.
How to Place an Order
Reference
- Dhiraj K., Ram H., et al. "DNA Bar-Code for Identification of Microbial Communities: A Mini-Review" EC Microbiology 2017, 219-224.
Our products and services are for research use only and cannot be used for any clinical purposes.
ITS Sequencing
