Supplementary Figure 4: Validation of findings of LOY using next-generation sequencing for six candidate subjects.

Low-coverage whole-genome sequencing was performed on 100 participants from the cohort. Among the 93 subjects with a median LRR in the male-specific region on chromosome Y (mLRR-Y, i.e., the median LRR for ∼2,560 SNP probes in the region chr. Y: 2,694,521–59,034,049, hg19/GRCh37) lower than –0.139 (i.e., the threshold for frequency estimation, Fig. 2), whole-genome sequencing was performed in 6 participants. Panel a shows LRR data from the male-specific region on chromosome Y (MSY) in these 6 subjects using box plots. The rightmost box (in all panels) contains data from the 94 sequenced individuals with an mLRR-Y value above the –0.139 threshold for frequency estimation. The red lines in all panels represent the expected normal state. Next-generation sequencing data from the 6 subjects and 94 controls are plotted in panel b. The median read depth in the MSY for the 94 subjects without LOY was 1.6 (s.d. = 0.6). The corresponding read depth in the six subjects with LOY was 1.3 (s.d. = 0.5). In comparison, the median read depth on chromosome 22 was 3.8 (s.d. = 1.4) in the 94 subjects without LOY and 3.8 (s.d. = 1.2) in the 6 subjects with LOY. Read depth data were used to estimate the ploidy of chromosome 22 and the MSY region on chromosome Y in comparison with the rest of the genome using FREEC software³⁹. The estimated ploidy is plotted in panels b and d. FREEC calculates ploidy for the regions of interest as the copy number value in each 5-kb window in the region of interest after GC content read count normalization, given a normal autosomal ploidy of 2. Panels c and d show that the copy number state on chromosome 22 is normal in the participants affected with LOY and is plotted in panels a and b, using SNP array and next-generation sequencing data, respectively.