DNA barcoding of fungal specimens using PacBio long-read high-throughput sequencing

Area of Science:

• Mycology
• Genomics
• Bioinformatics

Background:

• Molecular methods are crucial for identifying species lacking distinct morphological features.
• DNA barcoding of voucher specimens is standard practice in taxonomic and ecological research.
• Traditional Sanger sequencing has limitations in efficiency and cost for large-scale specimen identification.

Purpose of the Study:

• To compare the efficiency of long-read high throughput sequencing (HTS) with Sanger sequencing for fungal taxonomic identification.
• To evaluate HTS for detecting intraindividual gene polymorphism in fungal specimens.
• To assess the cost-effectiveness and success rate of PacBio HTS for DNA barcoding.

Main Methods:

• A workflow was developed using PacBio HTS on 423 basidiomycete specimens (195 species).
• Internal transcribed spacer (ITS) and 28S rRNA gene (LSU) markers were utilized.
• Data analysis focused on operational taxonomic unit (OTU) assignment and polymorphism detection.

Main Results:

• PacBio HTS demonstrated a higher success rate than Sanger sequencing at a comparable cost.
• Species identification was generally straightforward with dominant OTUs representing target organisms.
• PacBio HTS effectively detected widespread polymorphism within the ITS marker.

Conclusions:

• Multiplex DNA barcoding using PacBio HTS is a valuable, cost-effective tool for identifying numerous fungal voucher specimens.
• PacBio HTS accurately recovers alleles and paralogues, providing essential data for species delimitation and population genetics.
• This method enhances taxonomic identification efficiency and offers deeper genetic insights.