Nuri A. Temiz, Branden S. Moriarity, Natalie K. Wolf, Jesse D. Riordan, Adam J. Dupuy, David A. Largaespada, Aaron L. Sarver

Forward genetic screens using Sleeping Beauty (SB) mobilized T2/Onc transposons have been used to identify Common Insertion Sites (CIS) associated with tumor formation. Recurrent sites of transposon insertion are commonly identified using Ligation Mediated PCR (LM-PCR). Here, we use RNA-sequencing (RNA-seq) data to directly identify transcriptional events mediated by T2/Onc. Surprisingly, the majority (~80%) of LM-PCR identified junction fragments do not lead to observable changes in RNA transcripts. However, in CIS regions, direct transcriptional effects of transposon insertions are observed. We developed an automated method to systematically identify T2/Onc-genome RNA fusion sequences in RNA-seq data. RNA fusion based CIS were identified corresponding to both DNA based CIS (Cdkn2a, Mycl1, Nf2, Pten, Sema6d and Rere) and additional regions strongly associated with cancer that were not observed by LM-PCR (Myc, Akt1, Pth, Csf1r, Fgfr2, Wisp1, Map3k5 and Map4k3). In addition to calculating recurrent CIS, we also present complementary methods to identify potential driver events via determination of strongly supported fusions and fusions with large transcript level changes in the absence of multi-tumor recurrence. These methods independently identify CIS regions, and also point to cancer-associated genes like Braf. We anticipate RNA-seq analyses of tumors from forward genetic screens will become an efficient tool to identify causal events.