T2T Colletotrichum lini Genomes with Hifiasm: ONT R9 and R10 Read Processing and Assembly Guidelines for Fungi

The assembly of telomere-to-telomere (T2T) genomes is essential for understanding genomic architecture, especially in fungal pathogens with complex karyotypes, such as Colletotrichum lini, causing flax anthracnose disease. This study provides optimized guidelines for the T2T genome assembly using Oxford Nanopore Technologies (ONT) R9.4.1 and R10.4.1 sequencing data processed with the Hifiasm 0.25.0 assembler (with --ont module). We analyzed ONT sequencing data for four C. lini strains and compared basecalling tools (Guppy and Dorado), read filtration strategies (quality thresholds Q10/Q15 and length cut-offs 5 kb/10 kb), and genome coverage levels from 5× to 160×. Our results demonstrated that Dorado-basecalled reads consistently had higher average quality, especially the R10.4.1 data, leading to improved telomere resolution and complete mitochondrial genome assembly. Moderate genome coverage (40–65×) combined with Q15 quality and 5 kb length filtration for R10.4.1 data, or Q10 and 5 kb for R9.4.1 data, produced the most contiguous and complete assemblies. Overfiltration of reads by length and quality or conversely excessive coverage (>90×) reduced assembly quality, causing fragmentation or erroneous chromosome merging. With optimized parameters of ONT R9.4.1 and R10.4.1 sequencing data preprocessing, Hifiasm efficiently generated T2T and near-T2T assemblies of C. lini genomes: 53.7–56.1 Mb length, 13–30 contigs, 12–13 chromosomes (including 3–12 T2T chromosomes), complete mitochondrial genome, and >98.5% BUSCO completeness. These findings provide a solid framework for ONT-based fungal genome assembly, facilitating future research on genomic variation and pathogenicity in Colletotrichum and related genera.