Recent advances and challenges in <i>de novo</i> genome assembly

<italic>De novo</italic> genome assembly has entered the telomere-to-telomere (T2T) era, driven by breakthroughs in high-quality long-read sequencing technologies (PacBio HiFi and ONT UL), and advanced assembly algorithms. This review comprehensively examines the current state and challenges of genome assembly across various genomic contexts. We highlight significant achievements, including the construction of T2T reference genomes for multiple eukaryotic species, and the development of pangenomes incorporating diverse individual assemblies. However, several critical challenges persist: (1) the assembly of ultra-long, highly similar tandem repeats, particularly in rDNA regions; (2) haplotype-resolved assembly of complex polyploid genomes, especially autopolyploids; and (3) complete metagenome assembly and high-resolution binning. These challenges are compounded by the high costs and time-intensive manual curation required for current assembly workflows. To address these limitations, we identify key areas for methodological innovation, including: improved alignment algorithms for repetitive sequences, AI-driven assembly graph analysis, and enhanced metagenomic Hi-C binning techniques. The integration of these advancements will be crucial for achieving cost-effective, efficient, and scalable assembly of complete and accurate genomes across diverse biological contexts, enabling large-scale population studies and advancing our understanding of genomic complexity.