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bioRxiv · 10.1101/2025.05.23.655776

Sequencing the gaps: dark genomic regions persist in CHM13 despite long-read advances

Wadsworth, M. E.·Page, M. L.·Aguzzoli Heberle, B.·Miller, J. B.·Steely, C.·Ebbert, M. T. W.
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Abstract

Comprehensive genomic analysis is essential for advancing our understanding of human genetics and disease. However, short-read sequencing technologies are inherently limited in their ability to resolve highly repetitive, structurally complex, and low-mappability genomic regions, previously coined as "dark" regions. Long-read sequencing technologies, such as PacBio and Oxford Nanopore Technologies (ONT), offer improved resolution of these regions, yet they are not perfect. With the advent of the new Telomere-to-Telomere (T2T) CHM13 reference genome, exploring its effect on dark regions is prudent. In this study, we systematically analyze dark regions across four human genome references--HG19, HG38 (with and without alternate contigs), and CHM13--using both short- and long-read sequencing data. We found that dark regions increase as the reference becomes more complete, especially dark-by-MAPQ regions, but that long-read sequencing significantly reduces the number of dark regions in the genome, particularly within gene bodies. However, we identify potential alignment challenges in long-read data, such as centromeric regions. These findings highlight the importance of both reference genome selection and sequencing technology choice in achieving a truly comprehensive genomic analysis.

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Wadsworth, M. E., Page, M. L., Aguzzoli Heberle, B., Miller, J. B., Steely, C., Ebbert, M. T. W.. 2025-05-28. Sequencing the gaps: dark genomic regions persist in CHM13 despite long-read advances. https://doi.org/10.1101/2025.05.23.655776

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