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Old 05-09-2017, 06:04 AM   #1
frossit
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Default Focus error in Miseq

We started a MiSeq running and after the first cycle there was an error: "Best focus is too near edge of range: AutoFocus would have moved z to 0, outside soft limits os -0,295001144426642... -0,00499885557335775: ThroughFocusSacnCommand".
Is it a camera problem?
May I start the running againg?

Thanks in advance for your attention!
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Old 05-09-2017, 06:45 AM   #2
GenoMax
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Contact Illumina tech support and let them take a look at your sequencer.
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Old 05-09-2017, 07:35 AM   #3
thermophile
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There's no restarting a run with focus problems. It's unlikely to be a camera problem, more likely cluster failure, lens or z stage issue. Absolutely call tech support
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Old 03-20-2018, 09:08 AM   #4
Samm
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We have had this error several times as well. The run just stops without images to show whether there is under- or over-clustering. I am very doubtful if this is just a clustering issue. I think it is a hardware problem.
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Old 05-02-2018, 05:40 PM   #5
chuanwu
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I had the same issue twice in a row at Miseq platform. How did you guys resolve the problem. I called Illumina and they just questioned a lot about my library.

Thanks
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Old 05-03-2018, 05:04 PM   #6
ikripp
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I had the same issue - "Best focus too near edge of range" and after contacting tech support and being directed to run these tests:

Y Stage test
M3 mirror test
Z Stage test
Compensator test
Conduct volume test
Thermal Ramp test
Prime Reagent Lines
Optics test

I was told the run was significantly overclustered and to drop my loading concentration and reload.
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Old 05-03-2018, 05:25 PM   #7
chuanwu
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All the system checks passed.
The only issue for me why the quant of library measured by KAPA qPCR, Qubit, BioAnalyzer and LabChip GX are not consistent with each other. Which platform is reliable?
Thanks
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Old 05-04-2018, 03:00 AM   #8
gabrieltw
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KAPA is the most accurate, provided you calculate it correctly. We usually run librarys on BioA or AA to get the correct size distribution and use it to do the calculation with KAPA results.
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Old 05-11-2018, 05:27 PM   #9
chuanwu
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ikripp, have you had a successful run recently? What is your loading concentration with how much % of PhiX? Thanks
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Old 05-13-2018, 05:28 PM   #10
ikripp
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Quote:
Originally Posted by chuanwu View Post
ikripp, have you had a successful run recently? What is your loading concentration with how much % of PhiX? Thanks
We do approximately a run each week and vary rarely do we have what is considered an unsuccessful run. I base the loading concentration off of the pool concentrations as determined by a HSD1000 tape run on a tapestation 4200, again previous run pools. So its hard to say what loading concentration I have gone with but our standard PhiX is 10% which changes when we have a unique pool. I work with 16S amplicon pools for reference.
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Old 05-13-2018, 05:49 PM   #11
chuanwu
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What is the loading concentration ( ng/ul) you got based on tape station?
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Old 05-13-2018, 07:23 PM   #12
ikripp
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Quote:
Originally Posted by chuanwu View Post
What is the loading concentration ( ng/ul) you got based on tape station?
5pM made from the 20pM library as per Illumina protocol.
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