ITS rRNA Sequencing for Amplicon Sequencing

Introduction to ITS rRNA Sequencing

The Internal Transcribed Spacer (ITS) region of the nuclear ribosomal RNA (rRNA) cistron has emerged as the universal barcode for fungal identification. In mycobiome and microbial ecology research, amplicon sequencing targeting the ITS region provides unparalleled taxonomic resolution compared to other ribosomal markers. While 16S rRNA sequencing remains the gold standard for profiling bacterial communities, the structural diversity of fungi requires a highly variable genomic region to differentiate closely related species. This technical briefing details the biology, methodology, industrial applications, and future trends of ITS rRNA amplicon sequencing.

Biological Architecture & Primer Selection

The eukaryotic ribosomal cistron consists of the 18S, 5.8S, and 28S genes, separated by the ITS1 and ITS2 spacers. Understanding this structure is crucial for designing amplicon sequencing assays. Depending on the research goals, investigators typically target the ITS1 region, the ITS2 region, or the full-length ITS sequence (ITS1-5.8S-ITS2).

ITS1 vs. ITS2: Choosing the Right Target

Both ITS sub-regions have distinct advantages. Historically, ITS1 has been favored for studies targeting specific groups of fungi, such as Basidiomycetes, while ITS2 is often preferred for broad-spectrum fungal profiling because it exhibits less length variation and is less prone to PCR bias. In short-read sequencing platforms (like Illumina NovaSeq), researchers must choose one of these sub-regions because the entire ITS operon (~600 to 800 base pairs) exceeds the read-length limits of standard Next-Generation Sequencing (NGS) chemistry. However, with the advent of third-generation long-read sequencing (such as PacBio Sequel II and Oxford Nanopore PromethION), full-length ITS amplicon sequencing has become highly accessible, eliminating the need to choose between ITS1 and ITS2 and providing complete taxonomic profiles.

Primer Selection and PCR Bias

Selecting appropriate primer pairs is a critical factor in determining the success of an amplicon study. Classic primers such as ITS1-F, ITS2, ITS3, and ITS4 have been widely used, but they can show bias toward certain fungal phyla (e.g., Ascomycota vs. Basidiomycota). Modern assays employ degenerate primers or optimized primer cocktails to minimize taxonomic bias and ensure representative amplification of the entire fungal community within complex environmental samples.

Workflow of ITS Amplicon Sequencing

A standard ITS amplicon sequencing project involves several key stages, each requiring strict quality control to avoid contamination and ensure reproducible data:

• Sample Preparation & DNA Extraction: Fungal cell walls contain chitin and glucan networks that are highly resistant to lysis. Utilizing automated extraction systems like the TGuide Smart Magnetic Tissue DNA Kit ensures high-yield, high-purity genomic DNA extraction from challenging samples like soil, plant tissues, and stool.
• PCR Amplification: Target regions are amplified using barcoded primers. High-fidelity DNA polymerases are utilized to minimize sequencing artifacts and chimera formation.
• Library Construction & Sequencing: Purified amplicons are prepared for sequencing. High-throughput platforms like Illumina NovaSeq are ideal for deep profiling of short fragments, while PacBio Sequel II platforms enable full-length single-molecule real-time (SMRT) sequencing.
• Bioinformatics Processing: Raw reads are demultiplexed, quality-filtered, and clustered into Operational Taxonomic Units (OTUs) or resolved into Amplicon Sequence Variants (ASVs). Taxonomic assignment is performed using reference databases such as UNITE.