Resolution of ring chromosomes, Robertsonian translocations, and complex structural variants from long-read sequencing and telomere-to-telomere assembly

Abstract

Delineation of structural variants (SVs) at sequence resolution in highly repetitive genomic regions has long been intractable. The sequence properties, origins, and functional effects of classes of genomic rearrangements such as ring chromosomes and Robertsonian translocations thus remain unknown. To resolve these complex structures, we leveraged several recent milestones in the field, including (1) the emergence of long-read sequencing, (2) the gapless telomere-to-telomere (T2T) assembly, and (3) a tool (BigClipper) to discover chromosomal rearrangements from long reads. We applied these technologies across 13 cases with ring chromosomes, Robertsonian translocations, and complex SVs that were unresolved by short reads, followed by validation using optical genome mapping (OGM). Our analyses resolved 10 of 13 cases, including a Robertsonian translocation and all ring chromosomes. Multiple breakpoints were localized to genomic regions previously recalcitrant to sequencing such as acrocentric p-arms, ribosomal DNA arrays, and telomeric repeats, and involved complex structures such as a deletion-inversion and interchromosomal dispersed duplications. We further performed methylation profiling from long-read data to discover phased differential methylation in a gene promoter proximal to a ring fusion, suggesting a long-range position effect (LRPE) with heterochromatin spreading. Breakpoint sequences suggested mechanisms of SV formation such as microhomology-mediated and non-homologous end-joining, as well as non-allelic homologous recombination. These methods provide some of the first glimpses into the sequence resolution of Robertsonian translocations and illuminate the structural diversity of ring chromosomes and complex chromosomal rearrangements with implications for genome biology, prediction of LRPEs from integrated multi-omics technologies, and molecular diagnostics in rare disease cases.

Keywords: Robertsonian translocation; acrocentric p-arm; inversion; long-read sequencing; optical genome mapping; ring chromosome; telomere-to-telomere.

Graphical abstract

Figure 1

Structures of ring chromosomes (A) Nearly complete r(17) (NA10284). (B) r(17) with terminal p-arm deletion and breakpoint insertion of duplicated sequence from 11q12.3 (NA06047). (C) r(1) with terminal deletions as well as interstitial inversions and dispersed duplications. (D) r(13) with terminal q-arm deletions and breakpoint in the acrocentric p-arm. (E) r(14) with terminal q-arm deletion and breakpoint in the acrocentric p-arm. (F) r(15) with terminal and interstitial q-arm deletion and loss of most of the acrocentric p-arm. (G) r(21) with terminal q-arm deletion, inversion, and interstitial duplication. Left in each panel, derivative structure of the ring chromosome, with segment labels corresponding to the reference chromosome on the right. In (G), the gray connector between copies of segment 1 indicates uncertainty about their fusion breakpoints. Right in each panel, loess-smoothed estimated copy number of the corresponding reference chromosome. Below, segments relevant to each rearrangement are labeled. Red, deleted segment; blue, duplicated segment; cyan, retained acrocentric p-arm segment with unreliable copy number estimates due to repeat content; gray, segment with no change in copy number. Centromeres are shown as black ovals. Magnifying glasses depict segments, as zoomed-in views, that are too narrow to be visible in the main linear ideograms.

Figure 2

Structures of linear rearrangements (A) Complex pericentric inversion of chromosome 22 (DGAP234). Top, reference chromosome labeled with segments involved in the rearrangement, with cutouts showing IGV screenshots of soft-clipped molecules supporting each breakpoint. Bottom, derivative structure of the inverted chromosome. (B) Robertsonian translocation between chromosomes 14 and 15 (NA00479). Top, reference chromosomes labeled with segments involved in the rearrangement, with an IGV screenshot centered on segment 3 showing soft-clipped molecules supporting all three chromosomes involved in the translocation. Bottom, derivative structure of the translocation. Colored lines below the chromosomes indicate the source chromosome: light purple, chr14; yellow, chr15; dark purple, chr19. The dotted line around segment 5 indicates uncertainty regarding the location of its upstream breakpoint, which occurs in a satellite array spanning ∼8 Mb. Red, deleted segment; blue, duplicated segment; cyan, retained acrocentric p-arm segment with unreliable copy number estimates due to repeat content; gray, segment with no change in copy number. Centromeres are shown as black ovals. Segment sizes are not to scale.

Figure 3

Structure of a translocation with chromoanasynthesis between chrX and chr17 with an inserted duplication from chr2 (DGAP154) Top, reference chromosomes labeled with segments involved in the rearrangement. Bottom, derivative structures of the translocated chromosomes. Small segments are not to scale; see Table S3 for breakpoint coordinates. Colored lines below the chromosomes indicate the source chromosome: light purple, chrX; yellow, chr17; dark purple, chr2. Blue, duplicated segment; gray, segment with no change in copy number. Centromeres are shown as black ovals.

Figure 4

Allele-specific methylation proximal to the r(15) breakpoint of NA21885 Top, gene track showing exons and direction of transcription (A) Phased reads with methylated CpG sites marked with black circles and unmethylated CpG sites marked with open circles, where blue reads are from the unaffected haplotype and orange reads are from the ring haplotype. Below, gray vertical lines mark the conversion from “coordinate space” to “CpG space,” in which every position is a CpG site. (B) Orange and blue lines show the proportion of reads that were methylated at each site for the ring and unaffected haplotypes, respectively. (C) Smoothed depiction of the raw information from (B), in addition to gray lines showing the smoothed methylation fraction data from phased unaffected samples (see material and methods for details). The transparent blue rectangle highlights the differentially methylated region overlapping the transcription start site of ARRDC4. At right, the red dotted line marks the ring chromosome breakpoint.