[pmiguel]

I don't yet have first-hand experience with the Illumina workflow. But I would suspect that in cases where you are doing "quad" or "oct" runs on a SOLiD flow cell, bead prep is substantially more work than cluster generation on the Illumina. The process VanessaS describes above would be "x4" for a quad run.

The reagent costs for doing anything other than a full flow cell (E80) run on the EZBead make it not cost-effective compared to the manual method. Or, at least, of questionable cost-effectiveness.

VanessaS, do you do quad runs on your SOLiDs? If so, do you use the EZBead for them as well?
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Phillip

[PersianExcurzion]

Oh I know. My Old lab used the Illumina platform. If I had known SOLiD was this Labor intensive, I probably would not have taken this job...

Thanks for all the answers everyone!!

[pmiguel]

Oh I know. My Old lab used the Illumina platform. If I had known SOLiD was this Labor intensive, I probably would not have taken this job...

Thanks for all the answers everyone!!

Have you run the SOLiD yet? If not, don't be too bummed. I would think there are some advantages over the Illumina work flow. We just haven't done Illumina runs yet...

[DNADEB]

Has anyone had problems with the terminal transferase reagents? We have had only 1 successful run with our EZ bead system! No subsequent ones have worked and we do get results with the IKA and our old reagents. We were sent replacement kits twice and we are working with our THIRD lot!

[pmiguel]

A little more detail would be helpful. Terminal transferase does not occur on the EZ bead system, you have to do that after bead enrichment is complete. (You probably know that -- but if it escaped your notice, it would definitely cause you problems...)

That said, yes we have had problems. First, if you come in really low on your enrichment percentage -- unlike with the manual method, those beads are junk. I don't know where the cut off is. Maybe 8%? 5%? Do not attempt to use them. Just throw them out and start again.

Also, the problem we have with terminal transferase is that if we start with a lower amount of beads than is expected in the protocol we get serious clumping of our beads. I don't recall this being an issue until recently unless we were really low on the amount of beads being tailed.

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Phillip

[DNADEB]

No we have been told that we have had THREE bad lots of reagents -- term transf! How often would you expect this to happen?? Has anyone else?
As we have had only 1 -- the first-- good run on EZ bead, it is hard not to expect that there is something else wrong with the beads.

[pmiguel]

What did happen? By which I mean, you are saying you had three runs that were not good. But what was not good about them? What type of libraries were they? If you did WFAs, what were your titration metrics? What were your bead yields after enrichment?

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Phillip

[DNADEB]

We have done MANY controls and the only beads that stick to the slide are those that are done with the OLD reagents we had back in the freezer. All of the others fall off! The beads are the same beads and emulsions are done the same way. Only term transf from the old reagent box works! I am going to looking to beg some reagents from people -- maybe some I see at the ABRF meeting to get some that work.

[pmiguel]

What are your bead yields on the EZ Bead? If they are low, that can explain why the beads don't stick after tailing.

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Phillip

[pmiguel]

PS, I go into more detail on this issue here:

http://seqanswers.com/forums/showthread.php?t=8936

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Phillip

[DNADEB]

Bead yields are fine. Read your thread. BTW we were told that others are having this problem and sometimes the beads do not fall off until during the run!

[pmiguel]

We had our beads fall off on a non-EZBead run. Clogged storage buffer valve was leading to bubbles being pulled into the flowcell (through the FC gasket, it appears).

Still have psychological scars from that one.

We have seen weird tailing results recently, but they tend to suggest too much tailing, rather than too little. Maybe some of the terminal transferase that was supposed to be put in your lot, got put into ours?

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Phillip

[DNADEB]

As I said above, I am giving a talk on the SOLiD at a pre-meeting workshop at ABRF on Saturday and will be begging someone to give us some that works.

[pmiguel]

But the story is that those terminal transferase batches work fine with manual beads, but not with EZbeads? Sounds like a residual reagent issue from your Enricher, maybe? Have you tried doing an extra (manual) wash on beads you take off the Enricher and see if those will tail?

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Phillip

[DNADEB]

No ONLY the OLD reagent from the back of the freezer works! the new reagents do not work anywhere.
AND beads that did not work with the NEW reagents, can be MODIFIED with the OLD reagent and then they work!