A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2 Jianli Li, Hongzheng Dai, Yanming Feng, Jia Tang, Stella Chen,

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A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2 Jianli Li, Hongzheng Dai, Yanming Feng, Jia Tang, Stella Chen, Xia Tian, Elizabeth Gorman, Eric S. Schmitt, Terah A.A. Hansen, Jing Wang, Sharon E. Plon, Victor Wei Zhang, Lee-Jun C. Wong The Journal of Molecular Diagnostics Volume 17, Issue 5, Pages 545-553 (September 2015) DOI: 10.1016/j.jmoldx.2015.04.001 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Comprehensive workflow of sequence and copy number variation (CNV) analyses of PMS2. CNVs in PMS2 or PMS2CL are detected by both multiplex ligation-dependent probe amplification and capture-based next-generation sequencing (NGS). Long-range-PCR (LR-PCR)/NGS is used to differentiate the source of changes in PMS2 or PMS2CL. The LR-PCR/NGS is used for both sequence analysis and confirmation of CNVs in the active gene. Capture-based NGS is also used to overcome allele dropout. del/dup, deletion/duplication. The Journal of Molecular Diagnostics 2015 17, 545-553DOI: (10.1016/j.jmoldx.2015.04.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Identification of deletions in exons 1 to 10. A: Heterozygous deletion involving exons 5 to 9 in patient S2 was identified by coverage analysis of capture-based next-generation sequencing (NGS). The normalized NGS coverage generated a ratio of approximately 0.5 for deleted exons 5 to 9. B: The plot of mean coverage depth of the test sample (y axis) versus mean coverage depth of 20 reference samples, reveals exons 5 to 9 deletion as outliers (oval) below the 45° line, suggesting a heterozygous deletion. C: Deletion of exons 5 to 9 is shown by multiplex ligation-dependent probe amplification (MLPA) test. The ratios of probes targeted to exons 5 to 9 of PMS2 are approximately 0.5 in the MLPA test compared to normal reference DNA (two copies). D: A homozygous deletion of exon 10 is revealed by the ratio of 0 generated by probes targeted to exon 10 in MLPA test of sample S4. All error bars represent 2 SD. The Journal of Molecular Diagnostics 2015 17, 545-553DOI: (10.1016/j.jmoldx.2015.04.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Next-generation sequencing (NGS) coverage analysis of deletion/duplications and breakpoints. A: NGS coverage profiles of reference sample (R), three samples with exon 14 deletion (S6, S8, and C2), and one sample with duplication flanking exons 11 and 12 (S5). B: All deletions of exon 14 are confirmed by long-range PCR and gel electrophoresis. The deletion molecule of PMS2 has a band size of approximately 15 kb (bright band), whereas the nondeleted PMS2 allele and PMS2CL have a band size of approximately 16.7 kb (light band). C: Breakpoints of exon 14 deletion in sample C2 overlap with two Alu sequences. Blue and red arrows indicate the direction of Alu sequences. D: The sequence of breakpoints in patient C2 contains a 12-bp microhomology marked by solid black lines, and breakpoints are marked by red arrows. E: The breakpoints are marked by a red arrow in the alignment of two Alu sequences. The homologous sequence–mediated recombination in patient C2 and S6 is marked by a solid back line, and is marked by a dashed black line in patient S8. The Journal of Molecular Diagnostics 2015 17, 545-553DOI: (10.1016/j.jmoldx.2015.04.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Breakpoint analysis of case S5 with duplication involving exons 11 and 12. A: The sequence of the breakpoints is revealed by the adjacent mismatched sequences from next-generation sequencing. B: The breakpoint is confirmed by Sanger sequencing analysis. C: The primers used in nested PCR are marked with black arrowheads, and primers used for Sanger sequencing are marked with red arrowheads. The dashed box marks the region shown in A and B. D: The duplication is confirmed by long-range PCR followed by gel electrophoresis. F, forward; R, reverse. The Journal of Molecular Diagnostics 2015 17, 545-553DOI: (10.1016/j.jmoldx.2015.04.001) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions