Hello,
In C. elegans we are able to inject plasmid DNA into a worm and recover animals where the plasmids have linearized and assembled into an extrachromosomal array, which is heritably transmitted. We can then irradiate and recover animals where the extrachromosomal array has integrated into the genome. I have taken both of these steps and performed Illumina PE150 sequencing on a wt and several outcrossed lines carrying the integrated array. Through other means I have narrowed down the likely location of the integration site to the X chromosome between 4.5Mb and 10Mb. I found 3 candidate sites for the integration by looking for locations with a large number of single-mapped reads facing each other (suggesting the other read is inside the insertion). I am now trying to figure out which of these candidates contains the insertion. I've tried to assemble the insertions using MindTheGap and PopIns2, but neither was successful. Does anyone know of a good way to confirm the exact location of the insertion?
Some other complicating factors:
- I injected 3 plasmid simultaneously and all 3 are contained in the array/insertion. Tens to Hundreds of copies of the plasmids may be in the insertion (I made a "reference genome" out of one 1200bp segment of one of the plasmids and 1% of my reads mapped to it, while ~60% mapped to the worm reference genome).
- The linearization can happen anywhere in the plasmid, so the breakpoints will not be consistent at each site of plasmid attachment
- The plasmids contain several kb promoters from the worm genome, so many reads will have duplicate maps sites for the assembly and reference genomes
- The plasmids also contain E. coli DNA regions. As C. elegans food is E. coli, there is a large amount of E. coli DNA contaminating the NGS reads, so there are duplicate map sites here too.
Any thoughts would be much appreciated!
