Hi All,
These problems sound all too familiar. In our experience, 9 times out of 10
the problem can be traced to the array or an instrument related failure
requiring a service call. Some of our arrays have only lasted ~100 runs.
Part of this is probably due to the junk that we inject in our capillaries.
In our lab, a core facility that runs other peoples' samples with a varying
range of quality, we see a definite degradation of sequence read length over
time, even though the ABI standards work quite well. Sometimes regenerating
the array helps, but more often than not, we have to replace the array. I
think part of the blame lies with the samples themselves, but much of it has
to do with the variability of array quality. We've had plenty of problems
just getting an array that works. I think a testament to the whole picture
is how readily ABI will replace arrays without charge.
One of the ways we test our array's quality is by setting up pGEMs ourselves
and comparing them over time. In our hands, this will show an array problem
much earlier than the ABI standard.
Let's hope it's not the polymer lot...we have been using POP-6 lot 0101130
for the past 3 weeks and haven't noticed a problem.
David Mohr
DNA Analysis Facility
Johns Hopkins University
Baltimore, MD 21287
----------
>From: Thomas J Stelick <tjs11@cornell.edu>
>To: Recipients of ABRF List <abrf@aecom.yu.edu>
>Subject: Re: big dye lot?
>Date: Wed, Mar 14, 2001, 12:40 PM
>
> bob,
> the problem we have seems similiar. We had some real bad problems
> with a new array we installed in october. After beating our heads against
> the wall for a few months, we were able to get a confession that some of
> the arrays from abi had some problems. After we replaced the array in
> january, things started going better. But over time we have been seeing a
> slow degradation. A lot more noisy sequences and more outright
> failures. We have run the abi bid dye standard and the machine appears to
> be working correctly. (although if you have used these before the signal
> strengths are about 20 times the signal you get from a normal sequencing
> reaction). It has recently gotten much worse. We have either a bad lot of
> polymer, bad big dyes or a different chemical problem. It still might be
> an array issue though. We just did a regeneration and that did not seem to
> correct the problem. We are at about run 100 on this array. I have
> ordered in a different lot of big dyes to try that. Hopefully this helps
> and I can stop beating the wall.
>
>
>
>
>
> At 09:06 AM 3/14/01, you wrote:
>>Thomas J Stelick wrote:
>> >
>> > hello,
>> > We have been getting some inconsistant results on some of our sequencing
>> > and one problem may be the lot of big dye. The problem showed up around
>> > the time we started using a new lot. The lot # is 0101047. Is anyone else
>> > having problems. We are using a 3700. Or is anyone having a problem with
>> > pop-6 polymer lot # 0101130.
>>
>>
>>We have been questioning POP6 lot 0101130 ourselves. An instrument that was
>>working very well with read lengths of 700-800 suddenly changed to reads of
>>350-450 after only a POP6 lot switch. ABI had no other reports of a problem
>>with that lot.
>>
>>There were reasons to suspect other problems: perhaps a bad array (regenerate
>>array improved the situation and switching arrays got us up to 650 nt reads)
>>and perhaps an instrument problem (we started seeing strange drop-outs in the
>>array image data in caps 90-96).
>>
>>The symptoms after the POP lot change were (i) decreased resolution - broad
>>run-together peaks, (ii) *leading*-edges of peaks had reverse "tails" (did
>>that make any sense? I can't figure out how else to describe it) and (iii)
>>numerous capillaries suddenly gave no resolution at all. With such a routine
>>procedure as POP lot change, I can't image what we could have done that would
>>have toasted the array. I am still highly suspicious of the POP6 lot.
>>
>>To re-iterate, yes we were suspicious of POP6 Lot 0101130, but no, we could
>>not prove it was a bad lot, and it is currently giving us modest resolution
>>but NOT great. I would be very interested in hearing of others using that
>>lot of POP6 and what results they are achieving.
>>
>>Bob Lyons
>>University of Michigan
>
> Tom Stelick
> DNA Sequencing Facility manager
> Bioresource Center
> Cornell University
> Ithaca, NY 14853
> (607)254-4857
> http://brcweb.bio.cornell.edu
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