Figure 1.

Experimental approach. (a) Normal (N); primary (P) tumor samples; and metastatic (M) samples were collected. Sections were evaluated histologically to assess viability and composition prior to DNA extraction. (b) Transposon Insertion Profiling (TIP)-seq. A full-length (6kb) LINE-1 is diagrammed (blue). TSD, target site duplication; UTR, untranslated region; ORF, open reading frame. PCR amplicons initiate from an oligonucleotide complementary to the 3' UTR and extend to a restriction enzyme site. These are sheared and sequenced. Alignment to the reference genome shows read pileups (peaks, lower left). There are no alignments 5′ of the insertion; reads align to the plus (blue) and minus (orange) strands 3' of the insertion. Peaks found in neoplastic samples and not matched normal samples indicate potential somatic insertions. Reads spanning the polyadenylated tail and unique DNA indicate the point of insertion. Insertions are validated by spanning PCR (lower right). The gel shows four lanes with a marker in the leftmost; amplicons from normal (N), primary (P) tumor, and metastasis (M) are shown. The “empty” or pre-insertion site is amplified in all samples, whereas the insertion is recovered from primary and metastatic disease sites. (c) Structure of LINE-1 insertions. LINE-1 is frequently 5' truncated (upper left). Insertions can have inverted 5′ ends (upper right). The diagram shows a sequence breakpoint (//); the 3′ LINE-1 sequence is in the regular orientation (rightward arrow), and 5′ LINE-1 sequence is in the opposing orientation (leftward arrow). Finally, 3′ transduction events incorporate DNA downstream of the parental LINE-1 at the new insertion site.