Problems with gDNA prep for PacBio

[skerker]

Our goal is to prep genomic DNA from S. cerevisiae to make either a 10kbp or 20kbp library. We have been having trouble with sample quality (either contamination or damage?) resulting in short PacBio reads (only about 1-2kbp). The library "looks good" - it was the expected size ~10kbp - but something else must be wrong...

It seems that very high quality gDNA is needed for good PacBio results. If anyone has a protocol that they would be willing to share for fungi, I'd appreciate it!

We have tried, Qiagen and Zymo research products, in addition to old-school glass beads / phenol-chloroform extraction.

Best,
Jeffrey Skerker
UC Berkeley

[tacana]

Hello Jeffrey,
We have also had many instances in which our libraries look good but the results did not reflect that. How did the ZMW distribution looked?

In our case many of these samples had contamination issues and many were rescued using a Magbead cleanup. The protocol that we used to rescue the samples can be found on Pacbio's sample net.

http://www.smrtcommunity.com/Share/Protocol?id=a1q70000000HCu5AAG&strRecordTypeName=Protocol

Hope this helps

[skerker]

Thank you for the reply! You did magbead cleanup of the SMRTbell library? I have been submitting samples to a sequence center, so unfortunately I don't have much control over what gets done, or what steps failed. I am trying to get more information to help troubleshoot my experiment.

It seems to me that the original gDNA can be cleaned up further - using powerclean Pro kit form MoBio, or do additional cleanup of the final library.
Do you have a feeling for which approach is better?

The initial cleanup I can do - the latter would have to be done by the PacBio sequencing provider, but I could suggest that they do the additional cleanup step.

I found some posters from PacBio on the subject. I think they tried a few different cleanup methods (magbead, qiaquick, etc). So perhaps this is a common problem and should always be done as a final cleanup of the library before sequencing?

Are you part of a PacBio sequence center? If so, I could submit my samples to your center so at least I know they would be cleaned up if a problem arises? Or maybe suggest a center that you have had good success with?

Thanks again,

Jeff

[tacana]

Hi Jeff,
I sent you my answer in a private message. Let me know if you got it.

[MatDeclercq]

Hi Jeff, do you have a trace of the final library that is actually put on the SMRT cell?
Might be that there simply are too many small fragments, which preferentially load on the machine.
It's a bit of an obvious thing, since a seq center that regularly uses PacBio should know this and size select to retain only the longest fragments. But you never know..

[skerker]

Thanks for the reply. I'll check with the seq. center. We are trying again using gDNA that has been purified by a Qiagen Tip 20/G and then with powerclean from MoBio. Hopefully that will do the trick.

Jeff