Graph pangenome reveals functional, evolutionary, and phenotypic significance of human nonreference sequences

Abstract

Thousands of DNA sequences in global populations are not present in the human reference genome, named nonreference sequence (NRS). Long-read sequencing (LRS) technologies enable better discovery of NRS with large length, particularly in repetitive regions. Here, we de novo assembled 539 genomes in five genetically divergent human populations sequenced by LRS technology and identified 5.1 million NRSs. These NRSs were merged into 45,284 nonredundant NRSs, of which 66.2% were novel. 78.5% of NRSs were repeat sequences, such as VNTR and STR. 38.7% of NRSs were common in the five populations, 35.6% were population specific, while 21.3% were ancestral and present in nonhuman primates. 144 NRS hotspots spanned 141 Mb of the human genome and many NRSs contained known functional domains or intersected with coding genes. Based on graph-based pangenome, we detected 565 transcript expression quantitative trait loci on NRSs, of which 467 were novel. We also detected 39 NRS candidates for adaptive selection within the human population related to the language system and diabetes. GWAS revealed 14 NRSs significantly associated with eight phenotypes, such as anaemia. Furthermore, we identified 154 NRSs in strong linkage disequilibrium with 258 phenotype-associated SNPs in the GWAS catalogue. Our work expands the landscape of human NRS and provides novel insights into functions of NRS to facilitate evolutionary and biomedical research.