A Novel Method For High-throughput Sequencing Analysis Of Integration Sites

Integration of exotic DNA sequences into host genomes not only happens during the natural cycles of virus infection, but also occurs when viruses or transposons are used in biomedical researches. In addition, retroviral vectors are widely used in gene therapy and characterization of unintended insertions is necessary for the development of safer vectors and allows monitoring of possible genotoxicity. Integrome analysis quereis insertions on a genome-wide acale, which promises a better understanding in viral tumurgenesis, a more comprehensive tool for insertional mutagenesis screen and a better guideline for the development of integrating gene therapy vectors.The location of the insertions in the host genome is currently determined by PCR amplification followed by sequencing and mapping of the flanking genomic sequences. The Next Generation Sequencing technologies facilitate the identification of more inserts, and therefore more potential knowledge to add on related fields. Inverse-PCR, linker-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR) are employed widely in combination with454sequencing to identify integration sites. Alternatives like nonrestrictive LAM-PCR (nrLAM-PCR) and Mu-mediated method were developed to have more comprehensive genomic access. Although nrLAM-PCR has increased integration site retrieval over its antecessor LAM-PCR, its complex process including linear amplification and ssDNA ligation make it technically challenging. For the Mu-mediated method, the authors acknowledged that it could be challenging to obtain enough Mu integration events to cover the full human genome efficiently.We developed a massive anchored parallel sequencing (MAPS) approach to isolate and sequence integrants on the genome-scale level. MAPS used mechanical energy for DNA fragmentation, thus avoiding bias from restriction enzyme digestion and further providing overlapping tags for confident affirmation of the integration sites. It incorporates Illumina pair-end (PE) sequencing adapter as walking adapter to seam together LM-PCR with the Illumina’s PE sequencing technology. Using the example for the multiplex analysis of virus-host integrations in six pairs (cancer and adjacent tissues) of hepatocellular carcinoma (HCC) patients with hepatitis B virus (HBV) infections, we demonstrated that MAPS could isolate and characterize clonal populations of HBV integration events. In particular, we identified68insertions in the12HCC DNA samples in a single mulitplex analysis. MAPS is a universal, semi-quantitative and nonrestrictive method applicable for identifying any potential genome integration site.