Abstract Precision oncology depends on accurate read alignment, variant calling, and transcriptomic profiling - all of which inherit the biases of the reference genome used for analysis. Despite the availability of gapless Telomere-to-Telomere (T2T-CHM13) and Human Pangenome Reference Consortium (HPRC) assemblies, the field overwhelmingly relies on GRCh38, a reference that underrepresents global genomic diversity, particularly African diversity. We systematically evaluated ∼296. 5 Mb of African Pan-Genome (APG) contig sequences absent from GRCh38 to determine how much is recovered by modern references and whether the missing content is functionally relevant. While ∼40% of contigs aligned to T2T-CHM13 and ∼83% to at least one HPRC assembly, confirming them as authentic human sequence. Ancestry-associated mapping patterns, overlap with immune/HLA loci, OMIM genes and GWAS loci, demonstrate that missing reference sequence is not functionally trivial. Most notably, a subset of 742 contigs failed to map confidently to any current reference yet were not repeat-enriched and contained predicted coding and regulatory features with transcriptional evidence in two breast cancer cohorts. These findings demonstrate that reference bias is not merely a technical limitation but a mechanism by which clinically relevant genomic variation is systematically excluded from discovery, disproportionately affecting patients of African descent. Integrating ancestry-enriched sequence into population-aware reference frameworks is essential to improve variant detection, biomarker generalizability, and the equitable reach of precision oncology. Citation Format: Nyasha Chambwe. Gapless but Not Global: Ancestry-Enriched Functional Sequence Still Missing from Human Reference Genomes abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr SY38-01.
Nyasha Chambwe (Fri,) studied this question.