Gamaarachchi, Hasindu et al.
Complete telomere-to-telomere diploid genome assemblies have long posed significant challenges due to the intricate architecture of genomes. In diploid organisms, separating the two parental haplotypes becomes particularly difficult, especially amid highly repetitive sequences, centromeres, and telomeres. Traditional assembly techniques often leave gaps in these regions, undermining the ability to fully understand genomic structure and function. The complexity is further amplified by the need to accurately assemble stretches with limited sequence diversity, making a comprehensive, gap-free assembly a demanding goal in genomics.
To address these challenges, the iterative Cornetto sequencing and assembly protocol has been developed, leveraging a hybrid approach that combines PacBio HiFi sequencing with Oxford Nanopore Technologies (ONT) nanopore sequencing. This method capitalizes on the strengths of both technologies: the high base accuracy of PacBio HiFi reads and ONT’s extensive read lengths and adaptive sampling. The iterative nature of the protocol allows for refined assembly, ensuring that initially challenging regions are revisited and correctly sequenced. Notably, this approach is not limited to human genomes, and it can also be applied to other non-human vertebrate genomes enabling broad advancements in comparative genomics and evolutionary biology.
A critical component of Cornetto method is the use of the Hologic Diagenode Megaruptor 3 to produce long DNA fragments necessary for both long-read sequencing platforms. For PacBio sequencing, the Megaruptor 3 was set to generate fragments from 15 to 24 kb range, whereas for ONT sequencing, it yielded even longer fragments between 43–66 kb. These high-quality, long DNA fragments are essential for spanning complex genomic regions and ensuring the continuity and accuracy of the final assembly. By integrating the Megaruptor 3 into the protocol, researchers are able to achieve more complete diploid genome assemblies, paving the way for new insights into genome structure and function.
