Quantitative comparison of fungal genome assembly strategies using short and long reads from simulated and empirical sequencing data.

High-quality fungal reference genomes are essential for comparative, functional and evolutionary studies, yet fungal genome features such as repeats, structural rearrangements, accessory chromosomes and intron-rich genes can complicate genome assembly and the selection of cost-effective sequencing strategies. Here, we benchmark fungal genome assembly performance using simulated and empirical short- and long-reads datasets to evaluate how sequencing depth, assembler choice and genome characteristics influence contiguity, completeness, accuracy and computational requirements. Using simulated reads from complete fungal genomes spanning diverse sizes and compositions, we evaluated short-reads (SR), long-reads (LR), hybrid and polished LR assemblies across sequencing depths from 10X to 100X. Key trends were validated using empirical sequencing data from 10 fungal isolates assembled with multiple strategies, including different Flye assembler parameter sensitivity and SR polishing. Across datasets, LR produced the largest improvements in contiguity, with most gains achieved at ~20-40X coverage and diminishing returns beyond moderate depth. SR polishing substantially improved base-level accuracy at relatively low cost, with ~10-20X coverage often sufficient to approach maximal error reduction. Hybrid assemblers showed strong algorithmic variability, with trade-offs between contiguity, error rates and computational demand. Genome architecture also influenced outcomes, as larger and more feature-dense genomes benefited more from long-read data while GC content had limited impact. Overall, our results suggest that moderate long-read coverage (~30-40X) combined with modest short-read polishing (~10-20X), particularly using Flye plus Polypolish, provides a strong balance of contiguity, completeness, accuracy and resource efficiency for generating high-quality fungal genome assemblies.