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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.

Document Type

Article

Publication Date

2026

Notes/Citation Information

© The Author(s) 2026

Digital Object Identifier (DOI)

https://doi.org/10.1038/s41598-025-28283-0

Funding Information

This work was supported by the National Institutes of Health [R35GM138636, R01AG068331 to M.E.], the BrightFocus Foundation [A2020161S to M.E.], Alzheimer’s Association [2019-AARG- 644082 to M.E.], PhRMA Foundation [RSGTMT17 to M.E. and predoctoral fellowship to B.A.H].

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