Figure 1

Mobile Element Scanning (ME-Scan) Library Preparation and Sequencing Protocol. (A) dsDNA genomic DNA is extracted and then fragmented by sonication. An AluYb8/9 element is depicted (black rectangle: Alu element; gray box: Poly-A tail of the Alu; TSD: t arget s ite d uplication). Some fragments (darker) will contain most or all of the element along with some upstream genomic sequence. (B) Fragment ends are repaired, 3'A overhangs are added, and oligonucleotide adapters (pink) carrying sample-specific indexes (blue) are ligated onto the ends. (C) Multiple indexed libraries are pooled for subsequent processing. (D) A limited number of PCR cycles are performed using a biotinylated AluYb8/9-specific PCR primer (ALUBP2) and a primer (PEP2) that anneals to the adapters. PCR products in the 650-700 bp size range are selected using gel electrophoresis. (E) The biotinylated strands are purified away from other products using streptavidin-coated paramagnetic beads. (F) The biotinylated strands are amplified by PCR with primers matching the adapter sequences. The resulting product is checked using an Agilent Bioanalyzer DNA 1000 assay (electropherogram and gel-like image shown.) (G) Paired-end, 2x36-bp sequencing is carried out on the AluYb8/9-specific pooled fragment library using a custom Alu-specific primer (ALUSPv2) for the first (Alu junction) read and the standard adapter-specific primer (PESP2) for the second (genomic flank) read. The junction read begins inside the Alu element, yielding 16 bp of Alu sequence followed by 20 bp of genomic flank sequence. The flank read contains the 5-bp index and the 'T' added during sample preparation, followed by 30 bp of genomic sequence. Multiple read pairs are depicted, corresponding to different fragments carrying the same AluYb8/9 insertion (generic fragment diagrammed at bottom.)