3.5 WFA using 3.0 reagents?

[pmiguel]

Has anyone done a WFA (SOLiD trial ligation run for assessing the quality of a library) using an old 3.0 WFA reagent kit, but the new 3.5 ("3plus") flowcells/software? We just did one and got terrible results on a library that previously worked well. (New ePCR, same library).

We are going to try a new WFA and include a quad with left over beads that we know are good. But since we noticed that the WFA reagent tubes were drained dry by the autosampler, whereas we expected some liquid to be left over because the new, lower-profile, flow cells should require less reagents, I thought I would ask here.

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Phillip

[pmiguel]

The new WFA sucked also. The old beads we deposited (from an ePCR that had previously--back in v3 days--given us excellent titration metric) as a positive control gave poor results as well.

Has anyone run a WFA under v3.5?

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Phillip

[snetmcom]

wfa strip tubes are the same, no change.
ran three v3plus WFA's so far. all are fine.

what do your images look like?

[pmiguel]

wfa strip tubes are the same, no change.
ran three v3plus WFA's so far. all are fine.

Thanks. You are the first person to verify WFAs work under v3plus for me. Seems like a minor thing, but I could not get any one at AB to say anything other than they were unaware of any issues.

Now, were the WFA's you did with a 3.0 WFA kit? I know that it is said there is no difference between 3.0 WFA and 3.5 WFA. But I have no way of verifying that at this point.

what do your images look like?

Lots of off-axis beads. I would suspect that 100x too much library went into the ePCR, or something. Except that enrichment percentages were unremarkable (~15%) and we get the same WFA results (14-20% on axis) on a set of beads that previously were around 60% on axis.

There is no cheetahing. Although I'm not sure cheetahing would cause issues for v 3.5 since it is capable of dealing with very high density depositions.

Anyway, again, thanks for your response.

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Phillip

[snetmcom]

I would assume the WFA reagents have not changed. We used a v3 WFA strip tube and a newer ordered WFA box. The part number didnt change from v3 to v3 plus, that leads me to believe it's the same.

unfortunately, i'm not sure what else is going on in that slide of yours.

I was told there were no chemistry changes.