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Old 01-20-2011, 08:37 AM   #1
BL82
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Unhappy Problems with 454 GS FLX Titanium medium volume sequencing

Hi,

I've been running my first ever pyrosequencing reactions recently, using all the kit and instructions provided by Roche for the GS FLX Titanium series. We're using 8 regions per plate, so the medium volume reactions, and we're using MID primers, with 12 primers per region (96 samples per plate). Our first plate, which we sequenced earlier this week, seemed to come out ok; however, the second plate, which I'm currently running, doesn't seem to have worked. The run is still progressing, but at the moment on the screen it is reading "0" for all control reads, and very low numbers (in the tens) for sample reads - so, seemingly, the machine is having problems picking up the actual samples themselves.

At the moment, I'm unsure whether to repeat the emulsion stage fo the protocol, or whether it's likely to be a problem earlier on, e.g. with the PCR (I don't think so - but it's possible). I was unsure about the emulsion stage, because I had a yellow information sheet saying that the lot of emulsion oil we used may produce a clear layer on top of the emulsion, so it was difficult to tell if the emulsions were broken. However, I had reasonable return of enriched beads; well over the 340,000 needed per region.

I'm a bit confused about why one run has worked and another hasn't. Can anyone give me any tips about which part of the process this is likely to be a problem with?

Thanks!
BL
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Old 01-20-2011, 10:49 AM   #2
pmiguel
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When the run completes, look at the PPi flows with RunBrowser. (You may need to set the brightness maximum to 200 or so to see anything.)

If the PPi flow do have some signal, but the control and sample beads do not then you may have the same (undiagnosed) problem we had on a recent 454 run. Please let us know.

If the PPi flows are also blank then you probably have an instrument issue (eg, clogged valve, etc.)

The emulsion should not be at fault because your control beads are not key passing either.

--
Phillip

PS Our next run was fine...
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Old 01-21-2011, 12:43 AM   #3
BL82
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Hi there,

Thanks very much for this. I've had a quick look at the run program (although I'm not experienced at this!) and my whole plate is showing up as "no key", including the controls. I checked the individual nucleotide washes, and at the beginning of the run I was getting some coverage of Cs and Ts, but no As and Gs. After a few washes, the coverage for Cs and Ts gets very patchy, almost as if they've just been spilt on top of the plate instead of washed over it. By the end of the run, no nucleotides are showing up whatsoever.

As for PPi, at the beginning of the run, the whole plate is positive with intensity >1000 - and by the final wash, it is blank. I'm not really sure what this means. It seems like it could be an enzyme problem, but I'm not sure whether it would be a problem with the actual machine, or with the way I set up the sequencing run ... do you have any ideas?

Many thanks again!
BL
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Old 01-21-2011, 05:08 AM   #4
pmiguel
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You will want to contact support. (Here in the states they are called "GS-Support".) What you describe sounds likes a fluidics issue to me. Good luck. Please let us know what the final determination is.

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