SP‐PCR paired with NGS and bioinformatics analysis

The methods of DNA library preparation and bioinformatics analysis was the same as that previously described [11]. Briefly, the Covaris S220 sonication system (Covaris, Woburn, MA, USA) was used to shear 10 μg of genomic DNA from each mixed library with fragment sizes ranging from 200 to 400 base pairs (bp). Following the manufacturer's instructions, DNA fragments were purified using AMPure XP beads. NEBNext Ultra II, DNA Library Preparation Kit for Illumina (E7645; NEB, Beijing, China), was used to repair and add 3′dA overhangs of these fragments, then ligate with Splinkerette linker. The junction fragments of PB3′ and PB5′ ITRs were amplified in two consecutive SP‐PCR rounds to generate PB3 and PB5 libraries, respectively. These libraries were sequenced on a single lane with pair‐end reads of 2*125 bases using Illumina HiSeq2500 at BGI (BGI Tech, Shenzhen, China). For bioinformatics analysis, a fastx toolkit (https://hannonlab.cshl.edu/fastx_toolkit/) was used to trim the reads of adapters and PB tags and followed by mapping to the human reference genome (hg38) using bowtie 2 software (http://bowtie‐bio.sourceforge.net/bowtie2/index.shtml).