Christopher M. Watson, Nick Camm, Laura A

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Characterization and Genomic Localization of a SMAD4 Processed Pseudogene Christopher M. Watson, Nick Camm, Laura A. Crinnion, Agne Antanaviciute, Julian Adlard, Alexander F. Markham, Ian M. Carr, Ruth Charlton, David T. Bonthron The Journal of Molecular Diagnostics Volume 19, Issue 6, Pages 933-940 (November 2017) DOI: 10.1016/j.jmoldx.2017.08.002 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 SMAD4 Sashimi plots generated following the alignment of targeted capture data, using the RNA-seq aligner Star (https://github.com/alexdobin/star).11 The corresponding value of each arc records the number of reads crossing the reported splice junction. Alignment coverage data are displayed with y axis values ranging from 0 to 20,000 for sample 1, and from 0 to 6000 for all other samples. The Journal of Molecular Diagnostics 2017 19, 933-940DOI: (10.1016/j.jmoldx.2017.08.002) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 DNA sequence at the common SMAD4 processed gene integration site, located within SCAI intron 18 (https://www.ncbi.nlm.nih.gov/nucleotide; NM_173690.4). Genomic coordinates refer to human reference genome build hg19. A: The dashed line marks the breakpoint 5′ to the processed gene. B: The last nucleotide matching the SMAD4 3′ untranslated region is identified, immediately to the left of the dashed line. To the right of dashed line is a poly(A) sequence. C: The SCAI intron 18 integration site beyond the poly(A) tail. This sequence was generated using a reverse-strand primer. The four nucleotides located between the dashed lines are duplicated from the proximal breakpoint. The Journal of Molecular Diagnostics 2017 19, 933-940DOI: (10.1016/j.jmoldx.2017.08.002) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A schematic representation of the SCAI locus, displaying the exon arrangement of the SMAD4 processed pseudogene, which is consistent with that reported for NM_005359.5 (https://www.ncbi.nlm.nih.gov/nucleotide). Exons (green boxes) are drawn to scale using Gene Drawer (www.insilicase.com/Desktop/GeneDrawer.aspx, last accessed August 18, 2017). The Journal of Molecular Diagnostics 2017 19, 933-940DOI: (10.1016/j.jmoldx.2017.08.002) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Read coverage plots showing a distinctive cliff-edge profile at the boundaries of exonic coding sequences. Note also the reduction in read coverage between exons 6 and 7, due to gapped reads spanning intron 6. All samples are represented on a y axis scale representing 0 to 5500. Exon numbering is according to transcript NM_005359.5 (https://www.ncbi.nlm.nih.gov/nucleotide). All exons are drawn to scale, as are introns 3 and 6 (the remaining introns have been compressed to aid clarity). Blue boxes denote coding exons; green boxes denote 5′ and 3′ untranslated regions. The Journal of Molecular Diagnostics 2017 19, 933-940DOI: (10.1016/j.jmoldx.2017.08.002) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions