Covaris M220 and AmPure beads

[moerae]

Hi,

I'm to the NGS world and I just started my experiments the other day. I'm using a custom designed SureSelect XT capture kit and sheared my DNA using a Covaris M220. The protocol uses an S220/E220 but I don't have access to those machine and thought that the M220 would be all right. The settings that was in the SureSelect XT doesn't apply as one of the settings can't go that high on the M220. So I just sheared my DNA using the M220 protocol for fragmenting DNA to 150 bp as the SureSelect protocol wants fragments that are between 150 - 200 bp. I analysed the fragments on a Bioanalyzer high sensitivity DNA chip and found that the fragments are around 200 bp in size (the peak is between 220 - 300 bp on the Bioanalyzer). How important is the sheared DNA size and would this affect my downstream experiments?

Also, for the first AmPure bead extraction, part of the protocol suggest drying the pellet on a 37C heat block for 5 mins and not to let the pellet dry excessively as it affects elution. Someone changed my setting to 99C and I didn't realise and I over-dried my pellets. My elution is quite low for my samples... how much of an affect will this have on my downstream experiments?

Thank you

[moerae]

Can someone please please help? I'm really really stuck. If I carry on how likely am I to screw up the whole experiment?

[Skamp]

I have done a sequence capture experiment using another kit and I think the SureSelect works in a similar way. You create a whole genome shotgun library, incubate it with your probes, pull out the library fragments that bind to the probes and do a final clean up.

1. Does the size rang of the sheared DNA matter?

Probably, yes. If the SureSelect protocol uses AMPure XP beads to do the size selection, then yes the fragment size does matter because Agilent has adjusted the ratios of beads to keep a narrow size range during the library preparation step.

2. Is high incubation of AMPure beads bad?

Yes, if you over-dry your AMPure XP beads it is hard to elute the DNA off of them. This will result in less DNA for whatever you plan to do with it. Depending where you are in the protocol, this may have a big effect. Why don't you just try continuing with one or two samples and see how it goes?

[moerae]

Thanks for the reply. I'm about 50 bp out from what the protocol requires, and I've tried to ask Agilent but no one is replying to my email. I will probably just go on with the experiment and see what happens.

[mzahir89]

Hi moerae,

I have a similar question to yours. Can you kindly update what happened when you went on with the experiment? Did the excess 50-100bp have any effect?

[luc]

I assume longer inserts would havean increased tendency towards cross hybridizations; but I have no data to back this up.

[jlei_face]

I know this reply is super late but since it seems there are other posters with similar issues I'll put in my two cents:
When doing a library prep for first few times, it is recommended that you adhere to the supplied protocol as much as possible. Deviating even slightly (especially at the beginning) can have a huge impact on the end result and you won't know really how to troubleshoot.
Moerae: before beginning the actual prep, you should have done a few experiments with the covaris to obtain the proper fragment size. By slightly adjusting treatment time and duty factor you may have been able to find the correct settings for a 150-200bp fragment size.
As far as over-drying the beads, yes, this is a huge impact on your yield (as you saw). Decreased yield will also lead to lower sequencing diversity, maybe higher duplicates, etc. Given you knew what went wrong and how expensive sequencing is, I would not continue with this prep.