Copy Number Variation Sequencing for Comprehensive Diagnosis of Chromosome Disease Syndromes  Desheng Liang, Ying Peng, Weigang Lv, Linbei Deng, Yanghui Zhang, Haoxian Li, Pu Yang, Jianguang Zhang, Zhuo Song, Genming Xu, David S. Cram, Lingqian Wu  The Journal of Molecular Diagnostics  Volume 16, Issue 5, Pages 519-526 (September 2014) DOI: 10.1016/j.jmoldx.2014.05.002 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Performance of CNV-seq on low template DNA samples. Chromosome 4 CNV-seq profiles for duplicate 10- and 50-ng 46,XX and 46,XX,4p16.1-pter genomic DNA samples. CNV-seq profiles are log2 values of the normalized sequencing read densities (y axis) versus the number of sequential 5-kb sliding 60-kb sequencing bins (x axis). The upper [log2 (3/2)] and lower [log2 (1/2)] dashed lines indicate 100% (one copy) chromosome gain (duplication) and loss (deletion), respectively. The mean log2 value for CNV (blue tracking line), regions of repetitive sequences (red bars), and the centromere (black bars) are also shown. Input DNA of 50 ng was more optimal for accurate assessment of the 8.92-Mb 4p16.1-pter deletion (arrows). The Journal of Molecular Diagnostics 2014 16, 519-526DOI: (10.1016/j.jmoldx.2014.05.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Mate pair sequencing to confirm the breakpoints of the 0.22-Mb Xp22.2 and 0.08-Mb 6q26 deletions originally identified by CNV-seq. Mate pair sequence results of the 5-kb fragments spanning the deletion interval were used to define the approximate coordinates of the deleted regions relative to the hg19 reference genome and design PCR primers on either side of the breakpoints. Sanger sequencing of the bridging PCR products mapped both deletion sizes and breakpoints to the precise single nucleotide (arrow). The Xp22.2 and 6q26 deletions harbored by patients suspected to manifest X-linked Pelizaeus-Merzbacher disease and Parkinson disease were, therefore, confirmed by full and partial loss of PLP1 and PARK2 gene sequences, respectively. The Journal of Molecular Diagnostics 2014 16, 519-526DOI: (10.1016/j.jmoldx.2014.05.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Reproduciblity of CNV-seq for CNV. Triplicate CNV profiles showing the 0.22-Mb Xp22.2 and 0.08-Mb 6q26 deletions. CNV-seq profiles are log2 values of the normalized sequencing read densities (y axis) versus the number of sequential 5-kb sliding 60-kb sequencing bins (x axis). The upper [log2 (3/2)] and lower [log2 (1/2)] dashed lines indicate 100% (one copy) chromosome gain (duplication) and loss (deletion), respectively. The mean log2 value for CNV (blue tracking line), regions of repetitive sequences (red bars), and the centromere (black bars) are also shown. Both deletions (arrows) were reproducibly detected by CNV-seq in three independent replicate samples and showed the expected copy number change. The Journal of Molecular Diagnostics 2014 16, 519-526DOI: (10.1016/j.jmoldx.2014.05.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Examples of matching SNP array and CNV-seq profiles for four classical chromosome disease syndromes. The chromosome location and size of the CNVs identified by both methods was virtually identical. See Supplemental Table S1 for details of each CNV. The Journal of Molecular Diagnostics 2014 16, 519-526DOI: (10.1016/j.jmoldx.2014.05.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 CNV-seq diagnosis of ring chromsomes 14, 22, and 18. CNV-seq profiles and matching karotypes. CNV-seq profiles are log2 values of the normalized sequencing read densities (y axis) versus the number of sequential 5-kb sliding 60-kb sequencing bins (x axis). The upper [log2 (3/2)] and lower [log2 (1/2)] dashed lines indicate 100% (one copy) chromosome gain (duplication) and loss (deletion), respectively. The mean log2 value for CNV (blue tracking line), regions of repetitive sequences (red bars), and the centromere (black bars) are also shown. CNV-seq precisely mapped and sized the terminal breakpoints. The Journal of Molecular Diagnostics 2014 16, 519-526DOI: (10.1016/j.jmoldx.2014.05.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions