Improving Fungal ITS Sequencing Pipeline for the Characterization of the Respiratory Tract Mycobiome

The respiratory tract mycobiome, composed of diverse fungal communities, remains underexplored due to major methodological challenges. Unlike bacterial microbiome studies, which benefit from standardized protocols, fungal characterization still lacks consistency. Key limitations include the absence of fungal-specific DNA extraction kits and technical issues in next-generation sequencing (NGS), especially internal transcribed spacer (ITS) amplicon sequencing. Although ITS1 and ITS2 regions are recognized as markers for fungal identification, their application faces several hurdles: limited fungal annotation, primer mismatches causing incomplete sequence representation, and high variability in ITS copy numbers, complicating abundance estimates. These issues hinder accurate identification and quantification of fungal communities, particularly in respiratory samples. We hypothesize that optimizing DNA extraction, refining primer design, accounting for ITS variability and improving bioinformatics pipeline will enable a standardized workflow for reliable mycobiome analysis. To this end, a curated fungal mock community, including Candida albicans , Aspergillus fumigatus , Penicillium chrysogenum , and Malassezia furfur, will be used to develop and optimize the sequencing pipeline. The project will focus on: (i) optimizing extraction protocols to maximize yields across diverse morphologies (yeasts, spores, germ tubes); (ii) evaluating and refining ITS primers to improve specificity, addressing mismatches, polymorphisms, and secondary structures; and (iii) applying short- and long-read sequencing to produce high-quality genome assemblies, providing insights into ITS variability and copy numbers. Preliminary results indicate improved accuracy in detecting fungal diversity and quantifying community composition. The optimized pipeline will be validated on human respiratory tract samples and benchmarked against existing methods. By overcoming critical methodological gaps, this project aims to deliver a robust sequencing workflow for accurate respiratory mycobiome characterization. Publication History Article published online: 04 March 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany