SOLiD4 deposition problem

[twlin]

We just switch from 3 plus to 4 this year.
In the era of 3 plus, we don't have deposition problem until version 4. Lots of beads were washing off in the storage buffer washing step.

Synthetics beads (excellent deposition)along with our sample beads were test with brand new reagents include dep prep, XD depV2, XD storage buffer, XD slides.
Many our samples beads were washed off, except synthetic beads.

It seemed like problems rest in 3' modification.
However, we were told form AB that Bead Deposition kit(3 plus) and Bead Pre Deposition kit (4) are the same. The 3'mod protocols are exactly the same as well.

So is it because of the different slide?

Does anyone have similar problems. or any suggestion?

[RickC7]

SOLiD4 deposition problem

Hello,

I have not seen the same problem. I just completed my first WFA and started first full slide on v4 and beads are there. Just curious are you doing manual bead prep or EZBead? I used EZbead, set-up the 3'MOD at the end of the day and left it at 37C overnight.

Do you mean washing the slide with storage buffer right before putting the slide on the instrument? Strange that you would see the beads wash off at this point, but not when you pull new depo buffer through the chamber after incubating at room temp for 1 hour. did you spin the slide at 160G for 10 min?

Certainly sounds like 3'MOD issue. Try incubating overnight at 37C.

Good Luck
Richard

[twlin]

Hi Rick07,
Thanks for your replying.
Sorry I was not make our situation clear.
Un-deposited beads were washed off if depV2 buffer was pouring directly onto porthole, but they looked still "deposit" on the slide if pouring elsewhere onto the chamber. When washing beads with depV2 buffer by pipetting, sometimes beads are "moved". It will be more obvious in the case of un-deposited beads. And we did centrifuge for 10 min at indicated speed.

We do manual beads prep, and incubate at 37 for 2hrs in 3'mod step.
We tried 2nd round of 2hrs 3'mod, and overnight 3'mod on those beads.
They looked like no difference (color appeared on the slide) under naked eyes.

Our WFA worked fine though. 15 millions (nanodrop counts) input turned out to be 10 millions in WFA. Exactly the same beads were used for actual run. We didn't notice the deposition problem until that. (my guess is it may simply because total input beads are not that much in WFA.)

[pmiguel]

What kind of deposition numbers are you seeing on your run?

I think our results are similar to yours. We also do not have an EZBead system, so we did all those steps manually. However we did not really notice these un-anchored beads until we removed the slide from the flowcell after the run. Then we saw a large number of beads remained on the "mirror" surface of the flowcell.

We actually loaded 2x the number of beads we were hoping to see on the slide. We were nevertheless unable to reach 300,000 beads per panel said to be possible with v4. We saw a little over 250,000 beads per panel.

Our FAS warned us not to use detergent to wash the deposition chamber -- water only. Detergent apparently can interfere with deposition. So if you are seeing very low deposition numbers, that may be the issue.

One possibility would be that manual enrichment is not as stringent as EZBead enrichment and weakly templated beads do not bind well enough to the glass slide to remain there. But I am not hearing of anyone outside Foster City who is getting 300K/panel depositions so it seems unlikely this is just an EZBead vs. manual bead issue.

--
Phillip

[VanessaS]

We started our first v4 run on Thursday and have about 236K/panel = 550M usable beads. We were shooting for 600M, and accounted for 20% excess. For us this is better deposition than on the 3+, so far. We did get 500+M on the 3+ once, but generally closer to 400M usable beads, and we usually went with 40% excess, at least. I'm pretty happy with our first deposition, though I'd certainly like to have been spot on. We are going to try to deposit a little more next time and see what happens. We are using the EZ Bead system, and I gotta say, I'm in love!

[snetmcom]

Yeah I dunno what I was talking about.

[VanessaS]

3'mod should still be done at 37 degrees, but deposition is now done at RT, right?
I've only done one reaction on the EZ bead at this point, but the amount of time it saved me was mind blowing, and the quality and quantity of the beads as good as I get manually, which is typically a little over 70% best plus good beads. The E80 module gave us 1.7B beads with the WFA titration metric at 70%. Since I don't need 1.7B beads, I'm betting I can get close to 80% if I play with titrations.
What do you guys usually get for B+GB with the EZ bead system versus manual?

[twlin]

Hi All,
Our problems solved by AB field specialist.
Surprisingly, it was tubes we used that interfered with version 4 bead deposition. NO KIDDING.
Some unknown chemicals coating might ruin bead deposition on v4 but not on 3+.
Notably, using those tubes from lib prep to 3' mod step seemed work out fine as long as you don't use those from v2 dep washing step and the followings.
Not sure if we will deposit more beads by using other tubes from the beginning.
Anyhow, using tubes as recommended by AB is going to be a safe bet.

Hi Phillip,
Thanks for your reply.
The failed deposition gave 55k/panel.
Our successful one was 250k/panel while aiming 300k/panel.
We also observed some beads remained on the mirror surface of the flowcell even on the successful one.
So I am not quite sure is it going to be problematic.
The question I haven't figure out is that did beads stick on the flowcell being count as useful beads?

[lee]

Hi - what kind of tubes were you using? We have been using the Eppendorf low bind tubes. We are experiencing the same issues with our SOLiDs since the upgrade.

Thanks

[twlin]

DNA LoBind tubes from Eppendorf work fine but not Protein LoBind tubes.
That's what happened to us.
We don't want to waste those Protein LoBind tubes, so we still use them for ePCR, and then switch to DNA LoBind at the beginning of 3' modification.
Hope it helps,