Dan Russell

Nicholas Edgington
Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results.

Hey Nick,

We haven't tried that in any real experimental way. We do sometimes get samples with host DNA contamination, and in most cases it's relatively low coverage compared to the phage and thus isn't a problem. On the other hand, we have seen a few samples where the host coverage was high enough to drop the phage coverage to an unusable/undetectable level. So I guess: who knows?

Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help.

–Dan

mdgainey
Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem.
Maria

Dan Russell

mdgainey
Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem.
Maria

Hey Maria,

I don't really have any good tips to reduce the host DNA, but my sense is I'd try sequencing it and see what comes out. If the host DNA really is overwhelming, then it's back to the drawing board, but I think there's a decent chance the phage sequence would stand out from the bacterial chunks, and then you'd be done. So I'd just try prepping and sequencing without DNAse and see what comes out, then only worry about reducing the host DNA if that first attempt didn't work.

–Dan