Covaris shearing mixture of amplicons

[babine]

Hi,

I have a project in which I should sequence 16 loci in 96 individuals. Approximately half of the individuals are polyploids so the idea is to use 454 sequencing to avoid cloning. The only thing we didn't really consider is the size of the amplicons ranging between 700 and 2500 bp. A Covaris machine is available and we are planning to use it to shear the amplicons, but the question is: is it feasible to shear amplicons in a mixture (to reduce cost) when the ranging size is so wide?
Does anybody have experience with this kind of setting?

Thank you

[henry.wood]

I don't have much experience doing this, but I saw this paper http://www.ncbi.nlm.nih.gov/pubmed/22140562 which seems to address some of your concerns.

[babine]

Thanks for the paper!

But according to the person responsible for the 454 sequencing in my Institute, there should be no problem for the long fragments, but we might lose a lot of product in the short amplicons (<1 kb). Is that right?

[henry.wood]

I can't claim to be much of an expert, I was just passing on a paper that looked relevant. From my understanding the covaris doesn't over-shear too badly, so if you have the right settings, your smaller fragments will hopefully emerge intact. However it's at this stage my knowledge runs out, so I'll have to stand back and let others answer.

[Heisman]

You can test this easily by taking a couple of PCR products at different sizes and fragmenting them individually. Then run them out on a gel and see what the distributions are. If you do one fragment size per sonication it will be clear how they will perform.

[bbeitzel]

We have been using Covaris to shear mixed amplicons. There is no problem when the sizes are >~1kb, but smaller fragments require optimization of the Covaris parameters (in general they require a lot more time to be fragmented to the correct size range.) We usually have all amplicons > 1kb or all amplicons <1 kb, so I'm not sure what would happen if you had a mixture of the two sizes and used the small amplicon parameters to shear the mix. I would probably optmize conditions for a small amplicon, then mix in the larger amplicons and see what happens.

[babine]

Thank you for your inputs!

We will run a pilot experiment next week with 3 amplicons mixtures: 2 kb, 1 + 0,7 kb and all mixed under different time settings.
We will also consider running a pool of products with size <1kb and an other pool with the rest.

I should have more ideas by next week.
Hopefully it will work...

[danedwards]

I was wondering what progress you made with this? Were you able to optimize for the pooled size groups?