I´m mainly working on integration site analysis focusing on AAV-mitochondrial integration. So far I was using Cytoscape 2.8.3 to determine the CIS within the total DNA fraction isolated and sequencing data aligned to the whole human genome. In Cytoscape I used 50,000 as threshold of insertion sites (as Layout algorithm I´m using: Edge-Weighted Force-Directed BioLayout). As definition of CIS we used: a CIS of second order is defined by 2 integration sites (IS) within 30 kb, a CIS of third order by three IS within 50 kb, of fourth order by four IS within 100 kb and CIS of fifth, sixth or higher orders are defined by five, six or more IS within a window of 200 kb".
Now I also isolated mitochondrial DNA after AAV-infection of different cell types, and performed Linear-amplification mediated (LAM-)PCR to detect the AAV integration sites (sequences were aligned to the human mitochondrial genome). Now I´d like to use Cytoscape to detect the CIS (CIS order) in the mitochondrial DNA fraction as well. Therefore, I tested different thresholds (10, 100, 500, 1000, 5000) to figure out which might work best, as the mitochondrial genome has a size of 16,569 bp. So my question is how to determine the best threshold, as the results differ enormously between the tested thresholds (e.g. using 10 --> the highest CIS order is 3; using 1000 --> the highest CIS order is 39). Does anyone has an idea how to handle the issue with the mitochondrial genome?
Thanks a lot in advance!!!