Fig. 1

Hemi-specific PCR of P-element insertions. a Sequencing libraries were generated by nested hemi-specific PCR. First, asymmetric PCR enriches for P-element 3’ends using a P-element specific primer (P-enrich-F) that aligns to P-element from position 2752 to 2774 (out of 2907 total nucleotides). Next, a degenerate reverse primer is added recognize and amplify unknown sequences that are adjacent to P-element 3′ ends. Third, nested PCR with the P-nested-F primer cocktail (positions 2856 to 2877) and the degenerate reverse primer enhances PCR specificity for P-elements and produces amplicons with 5′ end read complexity, which is required for Illumina sequencing. Last, DNA fragments are amplified with indexing primers to allow for multiplexing. The resulting amplicons consist of adapters at each end, a P-element 3′ end and its adjacent genomic sequences. b PCR products from nested PCR with four degenerate primers (R4, R6, R10 and R11) are shown for two different annealing temperatures