Sanger Sequencers for Fungal Identification: Introduction, Appl

Introduction

Table of Contents

Sanger sequencing, developed by Frederick Sanger in 1977, is a chain-termination DNA sequencing method that remains the gold standard for molecular-level fungal identification.
It is widely used to determine the exact nucleotide sequence of specific fungal genes (e.g., ITS, D1/D2, LSU, SSU, β-tubulin, calmodulin) to accurately identify fungal species — especially those difficult to differentiate by morphology or culture alone.

A Qubit 4 Fluorometer (by Thermo Fisher Scientific, Invitrogen™)-Commonly used in molecular biology, genomics, and fungal DNA quantification workflows prior to PCR, qPCR, or sequencing (SangerNGS) — Fig. A Qubit 4 Fluorometer (by Thermo Fisher Scientific, Invitrogen™)-Commonly used in molecular biology, genomics, and fungal DNA quantification workflows before PCR, qPCR, or sequencing (Sanger/NGS)

Sanger sequencers operate based on capillary electrophoresis (CE) detection of fluorescently labeled dideoxynucleotides (ddNTPs), producing high-quality, single-read DNA sequences up to ~1000 bp.

Applications in Fungal Identification

1. Species-Level Identification

Targeted sequencing of the ITS region (Internal Transcribed Spacer) — the universal DNA barcode for fungi.
Confirms the identity of Candida, Aspergillus, Penicillium, Cryptococcus, and many environmental or opportunistic fungi.
Used for clinical mycology, taxonomy, and phylogenetic studies.

2. Confirmation of MALDI-TOF or PCR Results

Serves as a confirmatory test when phenotypic or MALDI-TOF identification is inconclusive.
Resolves cryptic species (e.g., Aspergillus fumigatus complex, Candida parapsilosis complex).

3. Antifungal Resistance Gene Analysis

Sequencing of ERG11, CYP51A, FKS, and CDR1/CDR2 genes to detect mutations responsible for azole or echinocandin resistance.

4. Phylogenetic and Epidemiological Studies

Enables strain typing and evolutionary analysis of clinical and environmental isolates.
Helps trace sources of hospital-acquired fungal outbreaks.

5. Reference Sequence Generation

Produces high-quality reference sequences for database submission (GenBank, UNITE, MycoBank).
Supports the development of fungal DNA barcode libraries for diagnostic use.

6. Quality Control and Teaching

Used in academic, diagnostic, and research laboratories for training in molecular mycology and sequencing.

Popular Sanger Sequencers

The box having the MinION sequencing device, Power adapter (FSP),Interchangeable international plugs- widely used in clinical mycology, genomics, and rapid pathogen surveillance — Fig. The box having the MinION sequencing device, Power adapter (FSP), and Interchangeable international plugs- widely used in clinical mycology, genomics, and rapid pathogen surveillance

ABI 3130/3130xl Genetic Analyzer
ABI 3500/3500xl Genetic Analyzer
ABI 3730/3730xl DNA Analyzer
SeqStudio Genetic Analyzer (Thermo Fisher Scientific)
CEQ 8000 (Beckman Coulter)

All these use capillary electrophoresis and fluorescent dye terminator chemistry for high-resolution sequencing.

Workflow Overview

DNA Extraction →
PCR Amplification (e.g., ITS1-ITS4 primers) →
PCR Product Purification →
Cycle Sequencing Reaction (ddNTPs) →
Capillary Electrophoresis (CE) →
Chromatogram Analysis (e.g., using Sequencing Analysis or BioEdit) →
BLAST Comparison (NCBI/UNITE databases)

Keynotes

Sanger sequencing remains the benchmark method for fungal DNA-based identification, offering >99% accuracy.
Particularly valuable for rare, slow-growing, or morphologically atypical fungi.
ITS region is the universal barcode; additional loci like β-tubulin, calmodulin, or LSU may be used for complex groups.
Produces a single read per locus, limiting throughput but ensuring high fidelity.
Cost-effective for small-scale testing; NGS is preferred for large sample sets.
Requires pure culture DNA and high-quality chromatograms for reliable identification.

Sanger Sequencers for Fungal Identification: Introduction, Application, and Keynotes