Small supernumerary marker chromosomes (sSMCs) remain a diagnostic challenge despite sequencing advances. As the field shifts toward cytogenomics, there is a need to establish methodologies to resolve these complex genetic variants at base pair resolution, as well as to identify their chromosomal origin and formation mechanism. Here, we apply long-read genome sequencing (lrGS) in combination with the telomere-to-telomere (T2T-CHM13) assembly to characterize the structure and genomic content of 10 clinically detected sSMCs. We use sequencing data to reconstruct the derivative chromosomes, identify breakpoint junctions (BPJs), and infer formation mechanisms. We resolve the BPJs of nine of the 10 sSMCs at base pair resolution. The analysis reveals six simple intrachromosomal rearrangements (one continuous and five discontinuous) with one to three BPJs, one complex three-way translocation with two BPJs, and two highly complex intrachromosomal rearrangements with five and nine BPJs, respectively. Breakpoint analysis reveals distinct mechanistic signatures: Simple sSMCs show features consistent with microhomology-mediated end joining (MMEJ) or microhomology-mediated break-induced replication (MMBIR), whereas complex sSMCs demonstrate evidence of translocation, chromoanasynthesis, and breakage-fusion-bridge (BFB) cycles. Haplotype analysis supports trisomy rescue in four cases, including all three complex sSMCs. In summary, our study demonstrates that lrGS combined with T2T-CHM13 enables detailed structural and mechanistic characterization of sSMCs, providing experimental support for disruption of trisomy rescue as a key formation mechanism. This work illustrates the feasibility of resolving highly challenging chromosomal abnormalities using long-read sequencing technologies.
Saether et al. (Wed,) studied this question.