ChildSeq-RNA The Journal of Molecular Diagnostics

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ChildSeq-RNA The Journal of Molecular Diagnostics Mohammed A. Qadir, Shing H. Zhan, Brian Kwok, Jeremy Bruestle, Becky Drees, Oana-Eugenia Popescu, Poul H. Sorensen The Journal of Molecular Diagnostics Volume 16, Issue 3, Pages 361-370 (May 2014) DOI: 10.1016/j.jmoldx.2014.01.002 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 RT-PCR validation of the newly identified EWSR1-ERG exon 7/exon 9 fusion transcript in the ES cell line COG-E-352. PCR products were amplified using primers spanning the breakpoint region of the EWSR1-ERG gene fusion, which was detected using ChildSeq-RNA, and then were separated by electrophoresis on a 1.2% agarose gel. Lane 1, Hi-Lo DNA marker (Bionexus, Oakland, CA); lane 2, the fusion-negative control cell line (HTC116) lacking ES fusion transcripts; lane 3, RT-PCR product (194 bp) for the EWSR1-ERG exon 7/exon 9 fusion transcript in COG-E-352 cells; lane 4, the nontemplate control; lanes 5 and 6, RT-PCR product (170 bp) for the control ACTB gene in the fusion-negative HCT116 and ES COG-E-352 cell line, respectively. The Journal of Molecular Diagnostics 2014 16, 361-370DOI: (10.1016/j.jmoldx.2014.01.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Detection of EWSR1-FLI1 fusion transcripts in fusion-positive serially diluted samples with control fusion-negative total RNA. Total RNA extracts from the ES cell line SK-N-MC were diluted with total RNA extracted from the fusion-negative cell line HCT116 at varying levels, as indicated by the x axis (100%, 50%, 10%, and 1% of SK-N-MC total RNA). The number of reads supporting the EWSR1-FLI1 transcript detected in each of the dilutions is shown on the y axis. The Journal of Molecular Diagnostics 2014 16, 361-370DOI: (10.1016/j.jmoldx.2014.01.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Correlation between gene expression measured using ChildSeq-RNA and whole-transcriptome sequencing (Illumina HiSeq2000) in clinical samples 5 and 6. Gene expression is defined as RPKM, and it is computed for each of the 12 genes targeted in the ChildSeq-RNA assay (ACTB, GAPDH, ERG, ETV6, EWSR1, FEV, FLI1, FOXO1, NTRK3, PAX3, PAX7, and WT1). The y axis denotes log2RPKM values derived from whole-transcriptome sequencing, whereas the x axis denotes log2RPKM values derived from ChildSeq-RNA. The dashed lines are lines of best fit (computed in log2 space). For clinical sample 5, the coefficient of determination using Spearman’s correlation (R2; computed in log2 space) was calculated to be 0.96, whereas for clinical sample 6, R2 was calculated to be 0.91. The Journal of Molecular Diagnostics 2014 16, 361-370DOI: (10.1016/j.jmoldx.2014.01.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions