v4 Deposition Efficiency?

[pmiguel]

To me it seems obvious that if you are running your SOLiD, you want to do so such that the maximum number of reads result. Obviously not at the expense of lower data quality, etc. But just as a general principle: increased numbers of reads for the same run chemistry costs == good.

Under v3+ we had great difficulty achieving the 500 million usable beads per slide metric until it was finally revealed to us that 500 million/slide was pushing the v3+ deposition method to the extreme. We had to deposit well over 1 billion beads to reach 500 million beads per slide.

So now we are nearing the time when we will be doing our first v4 depositions. New deposition procedure, said to give higher deposition efficiency but similar issue: we want to deposit the number of beads that will result in 700 million beads/slide, but not more. (Well, 710M or 720M would probably be okay.) The manual is no help at all in this matter and our FAS will only tell us to be conservative. You know, deposit 700 million beads and see what you get.

So, I am sure there are v4 runs that have been started at least. How about we post our real world "number of beads deposited" vs. "number of usable beads" results? This would all be done under the caveat that if you do go substantially over 700 million beads/slide, then you will likely have numerous panels will bead densities too high for the software to process.

That said, what, in your hands, did it take to get to x number of "usable beads" under v4?

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Phillip

[pmiguel]

Started our first v4 run. 1 billion beads deposited resulted in 508 million usable beads.

[snetmcom]

I take it this is based off p2 numbers on WFA?
how was the p1/p2 ratio?

Typically my variation in deposition has been isolated to WFA deposition problems. If you deposit your WFA poorly, then it translates directly to your full run. It's pretty easy to get variation in WFA bead numbers if you are not careful while preparing that slide. It's more likely you are depositing well under 1 billion... but you think it's 1 billion.

Is there a better way to count beads? not that i know, but i still use the color chart from time to time.

[pmiguel]

We use both the color chart and optical density measured on the nanodrop. We decided to deposit 1 billion beads, yes, because the WFA gave us about 70% deposition efficiency.

For these beads the WFA showed P2/P1 ratios of 88%. I don't put much stock in this metric. I remember under v2 it would nearly always report >100% P2/P1 ratios...

Yes, I think it is possible that our counting is off. But our Service Engineer always mentions finding beads in the waste lines, so I think substantial numbers get washed off the slide before the first ligation.

Much of this could have to do with the deposition aspect that AB is most secretive about: tailing -- and what the slide binding chemistry is as it relates to tailing. In the deposition procedure the tailing reaction went from 1 hr (v3+) to 2hrs (v4).

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Phillip