Tweet
Hi,
I’m working with an investigator who is interested in the encapsidation of host DNA in the SV40 virus. My main concern is how can I be sure that the host DNA that was sequenced was actually encapsidated and not just host DNA contamination? Is this feasible?
The virus samples have been prepared as follows:
African green monkey cells infected with SV40 virus, then disrupted after 48h of infection.
The crude extract was centrifuged; the virus pellet was resuspended and digested with DNAse I. The DNAse I resistant viruses were purified with a glycerol gradient then disrupted with EGTA and DTT, and the released minichromosomes were purified with a glycerol gradient.
Since the investigator is also interested in nucleosome positioning, they either sonicated the purified minichromosomes or digested the minchromosomes with micrococal nuclease, before sequencing on a MiSeq 75bp x 2.
When I align the reads to the SV40 genome and C.sabaeus genome using bowtie, the percentage of SV40 reads ranges from 48%-7% in 5 samples and the percentage of C.sabaeus reads ranges from 30% -49%.
Any suggestions or advice on how to approach this problem would be greatly appreciated!
I’m working with an investigator who is interested in the encapsidation of host DNA in the SV40 virus. My main concern is how can I be sure that the host DNA that was sequenced was actually encapsidated and not just host DNA contamination? Is this feasible?
The virus samples have been prepared as follows:
African green monkey cells infected with SV40 virus, then disrupted after 48h of infection.
The crude extract was centrifuged; the virus pellet was resuspended and digested with DNAse I. The DNAse I resistant viruses were purified with a glycerol gradient then disrupted with EGTA and DTT, and the released minichromosomes were purified with a glycerol gradient.
Since the investigator is also interested in nucleosome positioning, they either sonicated the purified minichromosomes or digested the minchromosomes with micrococal nuclease, before sequencing on a MiSeq 75bp x 2.
When I align the reads to the SV40 genome and C.sabaeus genome using bowtie, the percentage of SV40 reads ranges from 48%-7% in 5 samples and the percentage of C.sabaeus reads ranges from 30% -49%.
Any suggestions or advice on how to approach this problem would be greatly appreciated!