Unravelling ring chromosome structures and formation mechanisms by short-read and long-read genomic sequencing

Ring chromosomes (RCs) are rare cytogenetic abnormalities involving copy number variants (CNVs) and chromosome instability. Identifying the breakage-fusion sequences of RCs at nucleotide-level resolution can elucidate the cytogenomic rearrangements and ring formation mechanisms. This study employed short-read genomic sequencing (srGS) and long-read genomic sequencing (lrGS) alongside the telomere-to-telomere (T2T) reference genome to characterize the breakage-fusion events of 17 RC cases. Complete RCs without loss of euchromatin by a fusion of subtelomeric or telomeric regions were noted in a RC14 and a RC20. Incomplete RCs with intrachromosomal CNVs were noted in 15 cases, including a RC3, a RC4, four RC13s, a RC14, three RC18s, a RC21, three RC22s, and an RCY. srGS defined breakage-fusion sequences in single-copy sequences, and lrGS mapped subtelomeric and pericentric repetitive sequences using the T2T reference genome. The breakage-fusion sequences revealed ring formation mechanisms by intra-strand non-homology end-joining (NHEJ) in five RCs, microhomology-mediated end joining (MMEJ) in eight RCs, and microhomology-mediated break-induced replication (MMBIR) in four RCs. This study demonstrated the analytic validity and diagnostic utility of srGS and lrGS in delineating the genomic rearrangements in RCs for better interpreting clinico-cytogenomic correlations and further analysis of RC behavior in cell cycles.