Single-cell strand sequencing of a macaque genome reveals multiple nested inversions and breakpoint reuse during primate evolution

Abstract

Rhesus macaque is an Old World monkey that shared a common ancestor with human ∼25 Myr ago and is an important animal model for human disease studies. A deep understanding of its genetics is therefore required for both biomedical and evolutionary studies. Among structural variants, inversions represent a driving force in speciation and play an important role in disease predisposition. Here we generated a genome-wide map of inversions between human and macaque, combining single-cell strand sequencing with cytogenetics. We identified 375 total inversions between 859 bp and 92 Mbp, increasing by eightfold the number of previously reported inversions. Among these, 19 inversions flanked by segmental duplications overlap with recurrent copy number variants associated with neurocognitive disorders. Evolutionary analyses show that in 17 out of 19 cases, the Hominidae orientation of these disease-associated regions is always derived. This suggests that duplicated sequences likely played a fundamental role in generating inversions in humans and great apes, creating architectures that nowadays predispose these regions to disease-associated genetic instability. Finally, we identified 861 genes mapping at 156 inversions breakpoints, with some showing evidence of differential expression in human and macaque cell lines, thus highlighting candidates that might have contributed to the evolution of species-specific features. This study depicts the most accurate fine-scale map of inversions between human and macaque using a two-pronged integrative approach, such as single-cell strand sequencing and cytogenetics, and represents a valuable resource toward understanding of the biology and evolution of primate species.

Figure 1.

Genome-wide distribution of 375 inversions detected by Strand-seq between human and macaque genomes. Human chromosomes are shown on the left; orthologous macaque chromosomes, on the right. Orange lines between human and macaque ideograms show inversions detected by a simple strand switch. Green lines represent inversions within inversions, which are apparently direct by Strand-seq.

Figure 2.

Evolutionary history of two inversions. (A) Strand-seq view of Chromosome 13 shows the switch in orientation of a 2-Mbp region, suggesting the presence of an inversion (Chr13_inv1). The region was tested using FISH in interphase nuclei in multiple primate species and was inverted just in macaque, whereas all the other primates are in direct orientation similar to human. (HSA) Homo sapiens; (PTR) Pan troglodytes; (GGO) Gorilla gorilla; (PPY) Pongo pygmaeus; (MMU) Macaca mulatta; (CJA) Callithrix jacchus. (B) Strand-seq view of a 89-kbp inversion (Chr5_inv2) between BP1 and BP2 is shown. BES mapping and Illumina sequencing of primate clones show that the region is inverted in gorilla, orangutan, and macaque and is direct in chimpanzee.

Figure 3.

Evolutionary history and segmental duplication (SD) architecture of inverted region. (A) All inversions for which the evolutionary history has been determined are mapped on a phylogenetic tree in which the branch thickness is proportional to the number of inversions. (B) Inversions for which the lineage specificity has been determined are shown. The figure highlights the correlation between the presence of SDs at the inversion BPs and the size of the inversions.

Figure 4.

Comparison of chromatin structure and gene expression at a selected inversion (Chr18_inv4). Coordinates depicted are Chr 18: 9,140,001–13,490,000 (GRCh38). (A) Hi-C heatmap of human (top) and macaque (bottom) LCLs with predicted chromatin domains outlined in yellow, visualized in Juicebox. SDs are shown as colored blocks in the top track (taken from the UCSC Genome Browser). Genes are colored by differential expression: Red genes are up-regulated in macaque relative to human, blue genes are down-regulated, black genes are not differentially expressed, and gray genes were not tested. (B) The same locus is depicted with fibroblast Hi-C data. No differential expression analysis was conducted in fibroblasts.