Fig. 3

Expression and somatic retrotransposition of LINE-1 in secondary GBM. a. and b. LINE-1 ORF1p immunohistochemistry. a. Most primary GBMs and low grade gliomas do not have detectable LINE-1 ORF1p in this assay. Nuclei are counterstained in blue. b. About 74 % of secondary GBM cases are weakly, focally immunoreactive for LINE-1 ORF1p. (Brown). c-f. Identification of a somatically acquired LINE-1 insertion. c. The schematic depicts a plus (+) strand L1 as a rightward facing orange arrow with its 5′ inversion as a leftward facing blue arrow. The genomic LINE-1 sequence ends with a 3′ polyA tail. The gray right triangle illustrates the sequencing reads piling up (vertically, downward) when mapped against in the reference genome on the horizontal axis. d. TIPseq read alignments corresponding to an insertion detected in a secondary GBM tumor sample and not the patient’s blood DNA. The insertion is an intergenic LINE-1 on chromosome 17q22. Read depth is illustrated on the top (gray) and individual reads are stacked downward as blue and red bars denoting orientation. The greatest depth is immediately adjacent to the LINE-1 and extends 3′ of the element to create the triangular shape. e. An agarose gel electrophoresis of a validation PCR. The open arrowhead (lower) marks the pre-insertion allele and the solid arrowhead (upper) marks the amplicon that spans the LINE-1 insertion. The insertion is detected in the tumor (T) sample for this patient and not the corresponding blood cells (B). The LINE-1 is 5′ truncated at 1.8 kb. f. The annotated Sanger sequence for the LINE-1 insertion is shown in colored text: flanking unique genomic DNA (black), target site duplication (red), LINE-1 5′ inversion (blue), and LINE-1 3′ sequence and polyA tail (orange). Lowercase letters denote lower quality basecalls. These were confirmed by manually examining the trace file