The Physical Structure of the DUP4 Structural Variant
We initially decided to confirm this structural model by physical mapping of the DUP4 variant using fiber-FISH. We grew lymphoblastoid cells from the known DUP4 heterozygote from the 1000 Genomes project sample collection, HG02554, derived from a man with African ancestry from Barbados, and selected clones to act as fiber-FISH probes from the WIBR-2 human fosmid library spanning the region, based on fosmid end sequences previously mapped to the GRChr37 reference genome.
Fiber-FISH showed that the reference haplotype generates signals consistent with the genome reference sequence (Figure 1A). Because of the high sequence identity between the tandemly repeated glycophorin regions, there is extensive cross-hybridization of probes that map to the GYPB repeat with the GYPE and GYPA repeats. The GYPE repeat can be distinguished by hybridization of a small GYPE-repeat-specific PCR product, and the GYPA repeat can be identified by a gap in the green fosmid probe signal, caused by 16 kb of unique sequence in the GYPA repeat. Also, the overlap of the distal end of the blue fosmid probe with the proximal end of the GYPE repeat means that a small amount of blue signal at the distal end of the GYPB and GYPE repeats is detected, confirming repeat length and orientation. We identified DNA fibers showing an arrangement completely consistent with the DUP4 model proposed previously (Figure 1B).
Figure 1 Fiber FISH Analysis of the DUP4 Heterozygote Sample HG02554
(A) An example DNA fiber from the reference haplotype. The position and label color of the fosmid probes is indicated above the fiber on a representation of the human reference genome, and the interpretation of the FISH signals shown below the fiber.
(B) An example DNA fiber from the DUP4a haplotype. The Leffler model of the DUP4 haplotype is indicated above the fiber. The interpretation of the FISH signals shown below the fiber.
(C) An example DNA fiber from the DUP4b haplotype. The interpretation of the FISH signals is shown below the fiber.