Figure 2. Sequencing to validate the insertion and demonstrate recurrence.

(A) Illustrations of a reference Y chromosome, a reference X chromosome, and a Y chromosome with an X insertion. X specific sequence is indicated in red, Y specific sequence in blue, PAR1 reference sequence in purple, and LTR6B's in yellow. Arrows indicate primer pairs, with a bar representing an amplifiable product. The position of the SNPs of this study is shown in the order found in the amplicon. (B) PCRs using the Sanger.Junc primers shows bands for patients (P) and fathers (F), but not mothers (M), male controls (mc), female control (fc) or negative controls (neg), confirming the presence of an X specific insertional translocation in Y. (C) Sequenced amplicons of PCRs from part B, excluding reference upstream/downstream sequence. Red letters are from the X specific reference sequence. Yellow letters are from LTR6B reference sequence with red highlights indicating X specific LTR6B sequence and purple highlights indicating sequence specific for pseudoautosomal LTR6B. Purple letters indicate pseudoautosomal reference sequence. The gap underlined in red indicate bases missing from the X specific LTR6B. In black are annotated SNPs/Indels. In order from the beginning to the end of sequences, green boxes indicate SNP positions for rs2534625/rs12843082, rs2316283, rs2534627, and rs2857320. This Sanger sequencing identified two junction types, indicated as Junc1 and Junc2. (D) Phased haplotypes found through PacBio amplicon sequencing of the PacBio Duplication amplicons, with haplotypes assigned numbers indicated by gray boxes. Families in which both the patient and father were sequenced are color coded. No color indicates a sample in which the father was not sequenced. * Each individual has two haplotypes in the figure, except patients 10 and 15, which had a second unillustrated haplotype with many more variants that more closely resembled Y chromosome sequence.