Where we are in the T2T Genomes Era, Pt 2. T2T Sex Chromosomes
Following our previous DNA Day 2025 post covering where we are in the T2T (telomere-to-telomer) genomes era, we have a second expert Q&A covering the topic, this time from Kateryna Makova on the challenges (and opportunities) of assembly of T2T sex chromosomes.
Lets Talk about Sex Chromosomes
Kateryna Makova is the Verne M. Willaman Chair of Life Sciences and Professor in the Department of Biology in the Eberly College of Science at Pennsylvania State University, and has help lead efforts on T2T-sex chromosome assembly from the consortium. Kateryna and the consortium published the completed human Y-chromosome sequence in 2023, alongside presenting the gapless assemblies of the X and Y chromosomes of the great apes in 2024. Sex chromosomes have been particularly challenging to assemble due to their repetitive nature, dazzling diversity and incomplete reference assemblies, and these remained the last human chromosomes to be finished in humans. Until their completion containing many missing genes and regions of medical importance. With the Y chromosome well known to be important for human fertility, and the X chromosome harbouring many genes critical for reproduction, cognition and immunity. The huge improvements in T2T genome assembly finally providing new opportunities to study this previous “dark matter” of the genome.
Our T2T series has showcased such efforts with the publication of new computational methods for assembly of sex-limited chromosomes (see SRY: “Sorting long Reads of Y or other sex-limited chromosome”, published by Series Editor Jue Ruan). And our recent publication of a near-T2T short beak echidna genome, that investigated the evolutionary trajectory of (the extremely complex) monotreme sex chromosomes in unprecedented detail. While the first part of our Q&A on “where we are with T2T-genomes” had Guojie Zhang, Jue Ruan and Heng Li giving their benchmarking expertise, we thought in part 2 it would be useful to ask these same questions to an expert in the challenges of sex-chromosome assembly, and Kateryna has very kindly replied to these below.
The T2T Consortium paper on the complete sequence of a human genome was published 3 years ago. In that time “T2T” has become a buzzword for the complete genome references that have become increasingly ubiquitous not-just for humans, but other animal and plant species as well. Why do you think this move to sequence complete genomes has been so rapidly embraced, and do you feel “T2T” is a good label for the end products?
The telomere-to-telomere (T2T) genome assemblies represent an ultimate result of assembling the genome of an individual, this is why such assemblies have been rapidly embraced by the scientific community. Such assemblies provide information not only for the unique but also for the repetitive regions of the genome. T2T is a good label, however, it is frequently misused.
Over a decade ago the Assemblathon and Assemblathon 2 competitions highlighted the high degree of variability between genome assemblies, and helped kickstart more standardised ways to benchmark and assess reference genome quality. Do you think we are at a similar inflection point in genomics again, and what do you think are the minimal requirements necessary for a true T2T genome?
T2T genome assemblies should be the gold standard of assemblies—an assembly without gaps. In some cases, near T2T assemblies are acceptable, but they need to be marked as such. The acceptable regions to be finished in such assemblies in the future are telomeres and ribosomal DNA regions, for instance.
How big a problem is closing gaps, and what do you find as the best way to handle these?
It can be a big problem, based on our experience, and ideally gaps should be closed in the reference T2T genomes. This was particularly difficult to achieve for the human Y chromosome because it is so repetitive! For studying the intraspecific variation, nearly T2T assemblies might be sufficient.
Can you recommend any specific metrics for T2T genome quality, and are there any specific benchmarking methods that you recommend should be used for this purpose?
This is not my area of expertise, but our collaborators used multiple methods to validate the assemblies for the ape sex chromosomes, including coverage analysis, identification of erroneous k-mers, and assembly quality verification with Merqury. We also ran BUSCO analysis for great ape X chromosomes.
Now we have T2T-resolved human reference genomes (and a growing number of non-human ones); what is next for the field?
Having a human T2T reference is great, but one needs to remember that this is the genome of a single individual. I am the affiliate member of the HPRC (Human Pangenome Research) Consortium that works on sequencing and assembling the genomes of 350 humans representing multiple ethnicities. We need to generate such intraspecific data also for non-human primates. Such efforts involve collaborations across multiple disciplines, institutions, and countries.
Please check out our T2T series page to follow how these efforts progress:
https://academic.oup.com/gigascience/pages/t2t-series-closing-the-gaps-from-telomere-to-telomere
References
Nurk S et al. The complete sequence of a human genome. Science. 2022 Apr;376(6588):44-53. https://doi.org/10.1126/science.abj6987
Rhie A et al. The complete sequence of a human Y chromosome. Nature. 2023 Sep;621(7978):344-354. https://doi.org/10.1038/s41586-023-06457-y
Makova KD et al. The complete sequence and comparative analysis of ape sex chromosomes. Nature. 2024 Jun;630(8016):401-411. https://doi.org/10.1038/s41586-024-07473-2
Wang XB et al. An effective strategy for assembling the sex-limited chromosome. Gigascience. 2024 Jan 2;13:giae015. https://doi.org/10.1093/gigascience/giae015
Zhou Y et al. Chromosome-level echidna genome illuminates evolution of multiple sex chromosome system in monotremes. Gigascience. 2025 Jan 6;14:giae112. https://doi.org/10.1093/gigascience/giae112
