2025/05/28 by Mark E. Wadsworth, Madeline L. Page, Bernardo Aguzzoli Heberle +3 · 1 voice
Biochemistry, Genetics and Molecular Biology · #Cancer Genomics and Diagnostics #RNA modifications and cancer #Single-cell and spatial transcriptomics
paper · pdf · doi:10.1101/2025.05.23.655776
openalex publication_date 2025/05/28 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/14
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.