Inverted triplications formed by iterative template switches generate structural variant diversity at genomic disorder loci.

The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important pathogenic DNA mutation signature in genomic disorders and cancer genomes, its architecture remains unresolved. Here, we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the DNA of 24 patients identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted structural variant (SV) haplotypes. Using a combination of short-read genome sequencing (GS), long-read GS, optical genome mapping, and single-cell DNA template strand sequencing (strand-seq), the haplotype structure was resolved in 18 samples. The point of template switching in 4 samples was shown to be a segment of ∼2.2-5.5 kb of 100% nucleotide similarity within inverted repeat pairs. These data provide experimental evidence that inverted low-copy repeats act as recombinant substrates. This type of CGR can result in multiple conformers generating diverse SV haplotypes in susceptible dosage-sensitive loci.

Mind the gap: the relevance of the genome reference to resolve rare and pathogenic inversions.

Both long-read genome sequencing (lrGS) and the recently published Telomere to Telomere (T2T) reference genome provide increased coverage and resolution across repetitive regions promising heightened structural variant detection and improved mapping. Inversions (INV), intrachromosomal segments which are rotated 180° and inserted back into the same chromosome, are a class of structural variants particularly challenging to detect due to their copy-number neutral state and association with repetitive regions. Inversions represent about 1/20 of all balanced structural chromosome aberrations and can lead to disease by gene disruption or altering regulatory regions of dosage sensitive genes in cis . Here we remapped the genome data from six individuals carrying unsolved cytogenetically detected inversions. An INV6 and INV10 were resolved using GRCh38 and T2T-CHM13. Finally, an INV9 required optical genome mapping, de novo assembly of lrGS data and T2T-CHM13. This inversion disrupted intron 25 of EHMT1, confirming a diagnosis of Kleefstra syndrome 1 (MIM#610253). These three inversions, only mappable in specific references, prompted us to investigate the presence and population frequencies of differential reference regions (DRRs) between T2T-CHM13, GRCh37, GRCh38, the chimpanzee and bonobo, and hundreds of megabases of DRRs were identified. Our results emphasize the significance of the chosen reference genome and the added benefits of lrGS and optical genome mapping in solving rearrangements in challenging regions of the genome. This is particularly important for inversions and may impact clinical diagnostics.