Focus error in Miseq

[frossit]

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!

[GenoMax]

Contact Illumina tech support and let them take a look at your sequencer.

[thermophile]

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

[Samm]

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.

[chuanwu]

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

[ikripp]

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.

[chuanwu]

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

[gabrieltw]

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.

[chuanwu]

ikripp, have you had a successful run recently? What is your loading concentration with how much % of PhiX? Thanks

[ikripp]

Originally Posted by chuanwu

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.

[chuanwu]

What is the loading concentration ( ng/ul) you got based on tape station?

[ikripp]

Originally Posted by chuanwu

What is the loading concentration ( ng/ul) you got based on tape station?

5pM made from the 20pM library as per Illumina protocol.

[ArciMol]

Hi everyone!
Here I was searching about failed runs on MiSeq, and found this thread
In my case, we do runs on MiSeq each month, and recently we've had problems with 2 runs (MiSeq v3, 600c). In the first failed run, it was a problem of focus ("Best focus is too near edge of range(...)"); on the 2nd failed run, it has no clustering ("No usable signal found in the images"). I'll post lot number, in case it helps someone else:

MiSeq Reagent v3 600c box 1 of 2: LOT 20183241 and LOT 20270202
MiSeq Reagent v3 box 2 of 2: LOT 20178686 and LOT 20262956

We've also had the same answer from our local Illumina manager ... something like "it's your libraries fault" or "did you even load library on cartridge?". As we're no newbies, these answers doesn't help
Has someone else had failed runs with MiSeq, or reagents v3?

[ikripp]

Originally Posted by ArciMol

Hi everyone!
Here I was searching about failed runs on MiSeq, and found this thread
In my case, we do runs on MiSeq each month, and recently we've had problems with 2 runs (MiSeq v3, 600c). In the first failed run, it was a problem of focus ("Best focus is too near edge of range(...)"); on the 2nd failed run, it has no clustering ("No usable signal found in the images"). I'll post lot number, in case it helps someone else:

MiSeq Reagent v3 600c box 1 of 2: LOT 20183241 and LOT 20270202
MiSeq Reagent v3 box 2 of 2: LOT 20178686 and LOT 20262956

We've also had the same answer from our local Illumina manager ... something like "it's your libraries fault" or "did you even load library on cartridge?". As we're no newbies, these answers doesn't help
Has someone else had failed runs with MiSeq, or reagents v3?

My experience with "best focus" issues is that it does come down to an over-clustered run, although we have a very over-clustered run on the machine at the moment and it hasn't failed - it just has poor QC metrics. I have had runs with lower clustering (still over-clustered) that have failed yet this one has persisted.

I've never seen an error of no usable signal. What do the thumbnail images look like?

[ArciMol]

Originally Posted by ikripp

My experience with "best focus" issues is that it does come down to an over-clustered run, although we have a very over-clustered run on the machine at the moment and it hasn't failed - it just has poor QC metrics. I have had runs with lower clustering (still over-clustered) that have failed yet this one has persisted.

I've never seen an error of no usable signal. What do the thumbnail images look like?

Hi ikripp,
in the case of "no usable signal", we didn't even have thumbnail images, but we do have folder "Images", and there's a "Focus" -> "C1" folder, with .tif images file.
Here an example of name: FocusImage_0_exp_200_Focus T_IM_zpos_-0.15_method_CoarseFocus.tif

It looks like a huge black square. Literally no signal

[ppssvv]

Is a problem of focus alignment.
You need to realign the Focus position.