The Continuous Advancement of Reference Genomes

Reference genomes serve as the fundamental backbone for genomics and multi-omics data analysis, and high-quality reference genomes are pivotal for in-depth species-specific research. The evolution of reference genomes has undergone several key stages: from early individual genome drafts and chromosome-level scaffold assemblies to phased haplotype-resolved genomes, telomere-to-telomere (T2T) gap-free genomes, and high-resolution phased T2T genomes.

Beyond individual-level assemblies, the field has advanced toward population-scale pangenomes, which integrate genomic diversity across multiple individuals of a species, and the emerging concept of super-pangenomes, which encompass genomic variation across entire genera or higher taxonomic units.

Two core attributes define advanced reference genomes. The first is genome completeness, characterized by chromosome-scale continuity and gap-free coverage—a hallmark of T2T assemblies, which overcome the fragmented and gap-rich limitations of legacy reference genomes. The second is genomic coverage breadth, progressing from single-individual genomes to pangenomes and super-pangenomes.

Reliance on a single individual genome for species-wide genomic analysis can introduce significant reference bias, making pangenomes, with their integrated multi-sample genomic information, essential for capturing more comprehensive genetic diversity. Super-pangenomes further expand this scope, enabling the discovery of rare variants and complex genetic variation across broader taxonomic groups [1].

Recently, researchers have proposed the concept of an “ultimate reference genome,” defined as a phased telomere-to-telomere super-pangenome that maximizes the utility of genomic information and further highlights the critical value of T2T-level assemblies [2].

T2T Genomes: Definition and Advantages Over Legacy Assemblies

A T2T-level reference genome refers to a gap-free assembly that spans entire chromosomes from telomere to telomere, with no unresolved regions. For both individual reference genomes and pangenomic resources, assembly completeness is a critical prerequisite for robust genomic research.

In recent years, an increasing number of species have transitioned from gap-rich legacy reference genomes to high-quality T2T assemblies. Unlike their predecessors, T2T genomes resolve previously intractable genomic regions, including centromeres, telomeres, and other repetitive “dark regions” that were historically excluded or incompletely represented in genome assemblies.

This improvement enables the identification of novel genes and a broader spectrum of genetic variants, opening new opportunities for functional genomics and evolutionary research.

Ultra-Long Read Sequencing: A Key Driver of T2T Assembly

The development of T2T genomes has been directly enabled by advances in long-read sequencing technologies, particularly Oxford Nanopore Technologies (ONT) ultra-long read sequencing.

Conventional long-read sequencing, with an N50 read length of approximately 15–30 kb, is typically used for initial genome assembly and scaffolding. However, such approaches may still leave residual gaps when reads are unable to span large and complex repetitive regions.

In contrast, ultra-long read sequencing can generate reads with an N50 length of 100 kb or longer, allowing long and complex repetitive regions to be spanned more effectively. This capability provides direct, contiguous evidence for genome assembly, facilitates efficient gap closure, and supports the achievement of T2T-level completeness.

Therefore, ultra-long read sequencing has become a key technical component in the construction of high-quality T2T genomes.

BMKGENE T2T Genome Solutions and Success Case

BMKGENE offers a comprehensive workflow for T2T genome assembly, encompassing long-read library preparation and sequencing, Hi-C library preparation and sequencing, ultra-high-molecular-weight genomic DNA extraction, ultra-long fragment library preparation, high-accuracy SUP-mode basecalling, and specialized bioinformatics analysis for T2T genome construction.

A notable success case demonstrates the effectiveness of this approach. Li et al. utilized BMKGENE-generated PacBio HiFi reads (15 kb library) and ONT ultra-long reads (100 kb library) to assemble and close gaps in the red carrot genome (TXH4).

The resulting assembly achieved gap-free T2T-level completeness, comprising 9 chromosomes with a total size of 449.42 Mb, a contig N50 of 47.3 Mb, and a BUSCO completeness score of 98.7%.

This assembly represents the first T2T-level reference genome for carrot and provides an important resource for investigating the functional genetics of carotenoid and anthocyanin accumulation in this crop [3].

High-Quality DNA: Key to Ultra-Long Sequencing Success

The success of ultra-long read sequencing is highly dependent on the extraction of high-quality, ultra-high-molecular-weight DNA.

Fresh, healthy samples are an important prerequisite for successful DNA extraction. BMKGENE can provide recommendations on suitable tissue types and sample submission guidelines according to the characteristics of different species, helping maximize the likelihood of obtaining DNA that meets the requirements of ultra-long read sequencing.

References

1. He, Wenchuang, et al. (2025). The developments and prospects of plant super-pangenomes: Demands, approaches, and applications. Plant Communications, 6(2), 101230. DOI: 10.1016/j.xplc.2024.101230
2. Zhang, Jiahao, et al. (2025). Phased telomere-to-telomere super-pangenome: Definitive reference genome in plants. Trends in Plant Science. DOI: 10.1016/j.tplants.2025.11.002
3. Li, Xiao-Jie, et al. (2025). Analysis of telomere-to-telomere genome of red carrot TXH4 elucidates the role of DcLCYE and DcLCYB1 in lycopene accumulation in carrot. Horticulture Research, 12(11), uhaf192. DOI: 10.1093/hr/uhaf192