A T2T Benchmark Reveals How Reference Choice Shapes Human Genome Interpretation

Authors

Huang, Z., Chu, Y., Tian, Y., Shao, C., Wang, J., Zhang, X., Chen, J., Jia, Z., Li, L., Li, J., Lin, G., Zhang, K., Antonarakis, S. E., Kang, Y., Huang, J.

Abstract

The completion of telomere-to-telomere (T2T) human genomes has expanded the accessible landscape of human genetic variation, yet benchmark resources remain limited to conventional high-confidence regions defined by existing reference frameworks. Here, we generated a near-perfect diploid T2T genome (T2T-LIN) from a Chinese individual and established assembly-based truth sets by comparison with T2T-YAO, an ancestry-matched near-perfect T2T reference genome. The benchmark showed a heterozygous/homozygous SNV ratio of ~2, consistent with expectations under Hardy-Weinberg equilibrium, and enabled genome-wide evaluation of reference-dependent biases. We found that reference choice substantially influences genome interpretation: ancestry-matched linear T2T references provided the most faithful representation of individual genomic variation and enabled more accurate genome reconstruction than unmatched linear, diploid and graph-based references. Benchmarking previously inaccessible repetitive and structurally complex regions revealed substantial limitations of current variant callers that were masked by conventional metrics. The T2T-LIN and YAO-LIN benchmarks establish a T2T-era framework for evaluating reference-dependent genome interpretation and variant discovery across nearly the complete human genome.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 12 Sep 2026.

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