Abstract The aim of this study is to characterize the landscape of retrotransposon insertions in 202 colorectal cancer (CRC) patients to elucidate their role in colorectal tumorigenesis. Retrotransposons are genetic sequences that can copy themselves into an mRNA intermediate and insert elsewhere in the genome. Germline retrotranspositions can contribute to genetic variation however, high retrotransposon activity can also lead to genetic instability. Albeit retrotransposons are usually repressed in normal adult tissues, they become highly active in several cancer types, being most active in epithelial cancers such as CRC. Moreover, retrotransposon insertions have been difficult to detect with previous methodological approaches, which has resulted in very few genome-wide studies. Whole genome sequencing (WGS) was performed in 202 tumors and their corresponding normal tissue on the Illumina HiSeq 2000 platform; with 100 base-pairs paired-end reads. Each sample was sequenced to a minimum of 40x median coverage. To detect retrotransposon insertions we utilized TraFiC (Transposome Finder in Cancer). To identify retrotransposon-mediated transductions that were not identifiable using TraFiC, we utilized DELLY and the European database of L1-HS retrotransposon insertions in humans (euL1db). To characterize somatic insertions and decrease the rate of false positives, all insertions present in any of the normal genomes were filtered. After careful visual inspection of the paired-end read data, we estimated 80% of true somatic calls. We detected high retrotransposon activity in CRC with remarkable variation among patients. The mean number of insertions per tumor was 25, ranging from 0 to 197 insertions. In addition, we were able to identify few highly active retrotransposons that accounted for 70% of the retrotransposon-mediated transductions. We identified a total of 5065 somatic insertions, 2% of these insertions were located in exons, whereas 45% were in introns. We identified several genes with recurrent insertions, some of these loci being known fragile sites. However, we identified 13 known cancer genes with two or more insertions. Furthermore, we identified two patients with insertions in exon 16 of APC, suggesting that these insertions could be initiating tumorigenic events. To conclude, we found retrotransposon insertions to be a common phenomenon in CRC, contributing to genome instability and colorectal tumorigenesis. Citation Format: Tatiana Cajuso, Päivi Sulo, Riku Katainen, Ulrika Hänninen, Tomas Tanskanen, Johanna Kondelin, Jiri Hamberg, Niko Välimäki, Kimmo Palin, Outi Kilpivaara, Esa Pitkänen, Lauri A. Aaltonen. The mobile genome of colorectal cancer: Characterization of retrotransposon insertions in 202 colorectal cancer whole genomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4381. doi:10.1158/1538-7445.AM2017-4381