Multi-Marker Comparative Analysis of 18S, ITS1, and ITS2 Primers for Human Gut Mycobiome Profiling

Hiba Orsud¹Sumaya Hasan Zoughbor^1,2Fatima Al Dhaheri³Khalid Hajissa¹Manar Refaey⁴Suad Mohamed Ajab⁵Khaled Alswaider^1,2Nora Mohamed^1,2Obaid Alkaabi^1,2Zakeya Al Rasbi^1,2*

• ¹Department of Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates, AlAin, United Arab Emirates
• ²Zayed Centre for Health Sciences, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE, AlAin, United Arab Emirates
• ³Paediatric Department, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates., AlAin, United Arab Emirates
• ⁴Department of Genetics and Genomics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE, AlAin, United Arab Emirates
• ⁵Institute of Public Health, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE, AlAin, United Arab Emirates

Background: Gut fungi play crucial roles in human health. The profiling of human gut mycobiome continues to progress. However, adjustments in the selection of ribosomal DNA markers' regions can substantially affect taxonomic resolution of a population. In particular, the impact of using primers' combination is insufficiently defined. In this study, we investigated the performance of three targeted sequencing regions, ITS1, ITS2 and 18S rRNA, separately and in combination. Methods: Eight fecal samples from healthy individuals (n=4) and cancer patients (n=4) were selected as proof of principle for amplicon-based sequencing conducted with the DNBSEQ™ sequencing system. Quality-filtered reads were grouped into operational taxonomic units (OTUs) via USEARCH and categorized using the SILVA (18S) and UNITE (ITS) databases. Downstream bioinformatics encompassed diversity analyses, principal component analysis (PCA), and biomarker detection via LEfSe. To improve taxonomic coverage and compositional understanding, data were examined using ALDEx2 with CLR transformation, facilitating reliable differential abundance and effect size assessment in small sample metagenomic contexts. Results and Discussion: Among primers, the ITS2 and ITS1 enhanced the coverage of identified taxa with a quantity of operational taxonomic units of 183 and 158, respectively, compared to 58 OTUs of 18S. Accordingly, among primer combinations tested, the triple integration of ITS1-ITS2-18S produced the highest fungal richness, while the dual ITS1-ITS2 combined datasets enhanced the group discrimination analysis, showing the enrichment of Candida albicans, and scarcity of Penicillium sp. in cancer patients. Our findings based on ITS sequencing and the combination of ITS1 and ITS2 provide instructive information about the composition and dynamics of gut fungi in our initial test subjects, enhancing our understanding of their roles in gut homeostasis and the microbial shifts associated with cancer. Despite our approach being conducted with a limited cohort to establish methodological feasibility, it brings attention to multi-marker strategies, demonstrating that integrated primer datasets surpass traditional single-marker methods in both taxonomic coverage and biomarker detection sensitivity in low-biomass fecal samples. Our research provides a reliable starting point for future studies on gut mycobiome in both healthy and diseased individuals, which could lead to better diagnostics and treatments based on the microbiome profile.