Plasmid contamination in Long Tag Paired End library

[kmcarr]

Our core facility just did a long tag paired end library prep and sequencing run using the standard GS FLX kit and protocol. The sequence output looked fantastic but when I went to assemble it (gsAssembler v 2.0) a large fraction of the reads were rejected by the assembler. After a lot of head scratching and further analysis I discovered that 90% of the reads were plasmid DNA; that plasmid being some pBR derivative. Being on the bioinformtics side I didn't know the full details of the library prep but discovered the source of the problem after discussing it with the lab folks. For those who haven't done this protocol, at one step you are supposed to add 3 ug of pUC19 as a "carrier". You are supposed to separate your DNA of interest a few steps later via a biotin moiety on the paired end adaptor. Obviously our cleanup was not very efficient.

My question is, has anyone else experienced this plasmid carry over with the long tag protocol? If so what have you done to address the problem?

Thanks,

Kevin

[glacerda]

I am working with a 3K-LT run and I did not figure this problem before. Thanks for noticing that.
However, in my run, only 3% of the reads match a plasmid sequence.

[jpp]

Kevin,
We have got the same problem but a lesser extent, 50%, what is still very high. From Roche we have been told it was a bead washing problem, altough we followed strictly the protocol guidelines. In the eScholarship Repository, University of California we have found the following information, see the link http://repositories.cdlib.org/lbnl/LBNL-773E/ , where using Tween-20 to block the beads before DNA incubation disminish the inespecific pUC binding.
I hope it helps,we are going to try it the next time. Have you got rid off the pUC contamination?
Cheers

[kmcarr]

Thanks jpp. I had been directed to that poster by someone else as well and I passed the information along to our lab personnel (I'm bioinformatics and don't get my hands dirty at the bench ;-) ). We did some additional runs and I think they did implement some additional washing but I'm not sure if they used the method outlined in that poster. We ran three long tag paired end runs recently and still found some contamination but not nearly as bad. One sample was only a few percent but another was > 30%. I think the most significant factor effecting this was the amount of material we had to start with. We may tell customers if they want to do long tagged paired ends they will have to give us 15µg to start with.