From Fragmentation to Resolution: High-Fidelity Genome Assembly of Zancudomyces culisetae through Comparative Insights from PacBio, Nanopore, and Illumina Sequencing

Abstract Zancudomyces culisetae is an obligate symbiotic fungus inhabiting the digestive tracts of aquatic insect larvae, including black flies, midges, and mosquitoes. With a global distribution and high prevalence in disease-transmitting insects, Z. culisetae serves as a model for studying insect gut fungi. A previous draft genome assembly using Illumina short reads provided insights into its genome composition, such as a low GC ratio and evidence of horizontal gene transfer. However, its fragmented nature has limited deeper exploration of the evolutionary mechanisms shaping these gut symbionts. To address this gap, we generated a wealth of genomic resources for Z. culisetae using multiple sequencing platforms, including Illumina, Oxford Nanopore, PacBio-CLR (Complete Long Reads), and PacBio-HiFi (High Fidelity). This also provides an opportunity to compare these popular sequencing methods to suggest the optimal approach for fungal genome assembly. Our results suggest that PacBio-HiFi produced the most complete assembly, yielding a 27.8 Mb genome size with 26 contigs, representing the highest-quality genome of insect gut fungi to date. Additionally, we generated transcriptomic data to support genome annotation, identifying 8,484 protein-coding genes. Despite the improved genome quality, Z. culisetae lacks approximately 20% of Benchmarking Universal Single-Copy Orthologue (BUSCO) commonly found in fungi, reflecting adaptations to its obligate symbiotic lifestyle. This study not only provides valuable genomic resources for insect gut fungal research but also evaluates the strengths and limitations of current genome sequencing and assembly approaches, offering best practices for fungal genome analysis and genetic research. Article Summary Mosquito larvae harbor gut-dwelling fungi that may influence their development and disease transmission. Zancudomyces culisetae is a widespread fungal symbiont of mosquitoes, but its genome remained incomplete. This study explored and compared popular sequencing technologies, including Illumina, Oxford Nanopore, PacBio-CLR, and PacBio-HiFi, to generate a high-quality genome, alongside the first transcriptomic data. PacBio-HiFi produced the most complete assembly (27.8 Mb, 26 contigs) with 8,484 protein-coding genes, though ∼20% of core fungal genes were absent, reflecting symbiotic adaptation. This study establishes a genomic foundation for insect gut-dwelling fungi and evaluates available sequencing and assembly strategies for high-quality fungal genome production.