title: DNA library preparation for Illumina
design: Ten micrograms of genomic DNA from each mixed library was sheared by focused sonication with a fragment size of approximately 200 to 400 bp using the Covaris sonication system (Covaris S220). The fragmented DNA was purified using AMPure XP beads and analyzed on a 2% agarose gel to examine the size selection of the fragment. The fragmented DNA was distributed with a peak at approximately 300 bp. We used the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) to repair the ends of these fragments, and we added 3'dA overhangs and ligate with Splinkerette T-overhang linker (generated by annealing Splinkerette oligos SplkTp with SplkTm). Then, the junction fragments for the 3' and 5' ends of the PiggyBac transposon (PB3' ITR　and PB5' ITR) were amplified in two consecutive Splinkerette PCR rounds to generate PB3 and PB5 libraries for each pool. Approximately 750 ng of adaptor-ligated DNA was used in the subsequent junction-fragment enrichment. In the first round of PCR, a Splinkerette adaptor specific primer Sp-1 and primer PB5-1 or PB3-1 that was specific for the PB5’ ITR or PB3’ ITR, respectively, were used. The reaction volume was 25 μl, and the annealing temperature was 60°C for 30 PCR cycles. In the nested PCR round, 0.5 μl of the first-round PCR product was used as the template. Primer P5-Sp-2 contained terminal adapter sequences (P5) for Illumina solid-phase amplification and sequencing, and it also included a Splinkerette adaptor-specific sequence. Primer PB5-2.a or PB3-2.a contained sequences for Illumina sequencing and a specific sequence for the PB5' ITR or PB3' ITR accordingly. Other primers P7-PB-2.bn (n=1,2,3,4) contained terminal adapter sequences (P7) for Illumina solid-phase amplification, and a 6-base bar code followed by 13 base pairs which overlapped with the 5' terminal sequence of primers PB5-2.a or 3' counterpart PB3-2.a accordingly. The PCR reaction volume was 50 μl and the final concentration of three primers, P5-Sp-2, PB5-2.a (or PB3-2.a) and P7-PB-2.bn, was 1μM, 2 nM and 1 μM, respectively. The PCR cycling conditions were an initial annealing temperature of 50°C for 2 cycles, followed by an annealing temperature of 60°C for 18 cycles. Two Illumina DNA libraries of each HPBL based on PB3 and PB5 were generated, and a size selection of 300-500bp fragmented DNA was performed using AMPure XP beads following the manufacturer's instructions. These libraries were sequenced on a single lane of an Illumina HiSeq2000 device at BGI with pair-end reads of 2×100 bases read lengths following the manufacturer's recommended conditions. The customized PB5-ITR sequencing primer SP-read1 for read 1, Read2_primer for read 2 and an indexing sequencing primer Index_primer were used to decode the barcode of each library. Raw sequences were grouped into eight bins based on the barcode sequences incorporated into primer P7-PB-2.bn and each barcode library included two paired-end read files.