This course provides a comprehensive pipeline from genome survey to 3D structure analysis. The first part covers GenomeScope and Smudgeplot for K‑mer‑based estimation of genome size, heterozygosity, and repeat content, with detailed usage of Jellyfish/KMC and parameter tuning. The second part focuses on de novo assembly, covering Hifiasm (supporting HiFi, ultralong ONT, and Hi‑C data with phased outputs), 3D‑DNA (Hi‑C‑assisted error correction and chromosome anchoring), JBAT interactive manual curation (fixing misjoins, translocations, inversions), and ALLHiC tailored for polyploid genomes (pruning, partitioning, rescuing, optimizing, and building). Quality assessment includes GAEP, BUSCO (gene‑space completeness), Merqury (K‑mer‑based QV), and LAI (LTR integrity). The third part covers annotation: EDTA for automated TE annotation and library construction, and GeMoMa for homology‑ and RNA‑seq‑based gene prediction. The fourth part addresses Hi‑C 3D genomics, including HiC‑Pro quality control (valid interaction ratio), A/B compartment identification (hicPCA), TAD calling (hicFindTADs with insulation scores), and loop detection (hicHyperoptDetectLoops). The course emphasizes parameter optimization and result interpretation, offering systematic guidance for high‑quality genome assembly, annotation, and 3D functional studies.