Nanopore ultra-long sequencing and adaptive sampling spur plant complete telomere-to-telomere genome assembly.

The pursuit of complete telomere-to-telomere (T2T) genome assembly in plants, challenged by genomic complexity, has been advanced by Oxford Nanopore Technologies (ONT), which offers ultra-long, real-time sequencing. Despite its promise, sequencing length and gap filling remain significant challenges. This study optimized DNA extraction and library preparation, achieving DNA lengths exceeding 485 kb; average N50 read lengths of 80.57 kb, reaching up to 440 kb; and maximum reads of 5.83 Mb. Importantly, we demonstrated that combining ultra-long sequencing and adaptive sampling can effectively fill gaps during assembly, evidenced by successfully filling the remaining gaps of a near-complete Arabidopsis genome assembly and resolving the sequence of an unknown telomeric region in watermelon genome. Collectively, our strategies improve the feasibility of complete T2T genomic assemblies across various plant species, enhancing genome-based research in diverse fields. This study efficiently addresses the assembly of telomere-to-telomere (T2T) genomes in plants using the ultra-long sequencing of Oxford Nanopore Technologies. By optimizing DNA extraction and library preparation, ultra-long read lengths with an N50 of up to 440 kb was achieved. Combining ultra-long sequencing with adaptive sampling could effectively fill assembly gaps, facilitating comprehensive genomic research across various plant species. [ABSTRACT FROM AUTHOR]