ColoSeq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing Colin C. Pritchard, Christina Smith,

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ColoSeq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing Colin C. Pritchard, Christina Smith, Stephen J. Salipante, Ming K. Lee, Anne M. Thornton, Alex S. Nord, Cassandra Gulden, Sonia S. Kupfer, Elizabeth M. Swisher, Robin L. Bennett, Akiva P. Novetsky, Gail P. Jarvik, Olufunmilayo I. Olopade, Paul J. Goodfellow, Mary- Claire King, Jonathan F. Tait, Tom Walsh The Journal of Molecular Diagnostics Volume 14, Issue 4, Pages 357-366 (July 2012) DOI: 10.1016/j.jmoldx.2012.03.002 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 ColoSeq workflow. The Journal of Molecular Diagnostics 2012 14, 357-366DOI: (10.1016/j.jmoldx.2012.03.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Depth of coverage. Depth of coverage from a representative patient sample run on a single lane of a HiSeq2000 instrument in a 96-plexed ColoSeq run. A: Distribution of coverage for each targeted base. Each bar represents the number of base pairs (in 1000s) at a particular depth of coverage. Of the targeted bases, 98.5% have a minimum of 50-fold coverage. B: Median coverage across each ColoSeq gene is shown (average coverage of 475-fold). Areas with depth of coverage >1000-fold reflect capture of highly homologous genomic regions. The Journal of Molecular Diagnostics 2012 14, 357-366DOI: (10.1016/j.jmoldx.2012.03.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Deletions and duplications detected by normalized depth of coverage. Three examples of genomic deletions (MLH1 exons 14-15, MSH2 exons 1-6, and PMS2 exon 8) and one example of a genomic duplication (MLH1 exons 6-12) detected by ColoSeq. Read depth is normalized across the 96 samples on a run. Significant deviations less than or greater than a normalized ratio of 1 across adjacent baited regions reflect genomic deletions (red) and duplications (blue). Genomic coordinates shown on the x axis are hg19. The Journal of Molecular Diagnostics 2012 14, 357-366DOI: (10.1016/j.jmoldx.2012.03.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Between-run and within-run reproducibility. Between-run and within-run reproducibility are shown as a function of minimum threshold for indel variants and calling single nucleotide (SNV). Variants were considered reproducible if they met or exceeded the specified cutoff value in the index sample and appeared in the replicate sample regardless of cutoff value. Reproducibility is shown for all variants in single-copy regions of exons and splice junctions. A threshold of 15 variant reads was chosen for the assay. Black squares, between-run; grey circles, within run. The Journal of Molecular Diagnostics 2012 14, 357-366DOI: (10.1016/j.jmoldx.2012.03.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions