We have worked out the whole genome sequence of a virus by the traditional sequencing methods (shotgun library). The size of the circular-form of the genome after sequencing is 108kb, whereas initially when we did an electrophoretic mobility assay it showed a band at 125kb. I would like to know what reasons the difference can be attributed to.
Thanks. We have conducted PFGE to get the viral DNA at 125kb. We also employed the EcoRI and methylase treatment for the digestion, but got a single striking band at 125kb, that seems to assure the stability of the genomic DNA. After cloning and shotgun sequencing, the circular genome reads 108 kb, that seems appropriate to us after genomic comparisons. The question is whether the distance of migration by PFGE depends on agarose gel concentration? Also whether partial digestion by restriction enzymes are always necessary to separate the DNA by PFGE? What will be the problem if the viral genome is in its circular form?
The viral DNA won't leave the plug unless it's linearized. If you don't have any inverted or other forms of repeats in the genome, I would tend to trust the assembly. Try mapping your raw reads back on to the assembled genome and look for non-specific matches or other aberrations.
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