gDNA fragmentation with Covaris

[arvi8689]

i am planning to do some exome sequencing and now I am working on standardizing the covaris protocol to get a gDNA complex size range around 225 basepairs.

I get a good peak at 225 basepairs and a lot of DNA within the region i want but there is some amount of DNA outside the range which are either too small or too big. Should I just ignore it or do I need to do a size selection by running a gel. I am skeptical to do that because of the yield issues during gel purification.

Broad uses covaris and does not do size selection by gel purification and they are very confident because they get a very accurate peak. I am not sure if my peak is that accurate. I am attaching the results of my bioanalyzer run. Can some one help me with this and check if its a nice peak to go ahead with it?

Thanks

[jorgemon]

Hi Arvi8689.
Your peak looks like very good.
We don't do size selection by gel because we are doing a purification with Ampure Beads XP after fragmentation, then you lose all small fragments down 100bp. For the big fragments you can refragment the DNA some minutes more (5 for example). Then you will see that the peak is more narrow and compact and that big fragments will disappear. So I would go ahead with it, but if you want a peak more narrow, you need refragment that gDNA.
Regards

[TonyBrooks]

We fragment for a relatively long period (6 minutes) in line with the Agilent SureSelect Exome protocol. When using Ampure XP beads all through the protocol (1.8X reaction volume) we notice that we remove fragments <200bp and >400bp by the end of the protocol. If you want a really tight size selection, you can't beat a gel excision.
I would be tempted to carry on with the protocol and if you still want a tighter library size, then gel excise at the end. We typically see yields of >300ng at the end of our enrichments. Even if you lose 90% of that, you'll still have buckets of library for sequencing.