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Old 09-22-2015, 11:21 AM   #1
thermophile
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Default MiSeq runs failed "focus out of spec"

I'd like to take an informal poll. How many of you that routinely run a MiSeq have had multiple runs fail because focus is out of spec or Z-axis out of spec. I took over running a MiSeq in Jan, since then I've had 9 runs fail because of focusing issues, most times 200+ cycles into the run.

We typically run 500 cycle kits and have had this issue on both high (genomes) and low (amplicon) complexity samples. Yes Illumina replaces the kit and dispatches a tech but every time we loose a week of machine time. I've talked to a couple of people IRL who have MiSeq and neither have ever had a run fail because of focus. Several times it's seemed that "the" cause of the issue was found-cables rubbing on the stage, etc. But the failures keep happening.
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Old 09-22-2015, 12:51 PM   #2
cheezemeister
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We've probably done fifteen runs since getting our MiSeq and two have failed because of focus issues during cycle 1. Tech support tells us that it's because of either underclustering or overclustering of the cell, but without an image taken we cannot prove which, if either. I am skeptical that clustering problems are the cause, since both times this has happened we simply loaded a new flow cell with no changes and the run worked fine. Illumina replaced the kits but, as you mentioned, we lose time.

Generally, I'm not too impressed with the MiSeq. They're big on marketing the 300-bp read length, but I've found that the quality drops off quite dramatically after about 250-260 bp, such that our read merging starts to tank. They say they're working on improving this, but that doesn't help me right now. Improvements could be released tomorrow, or never.
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Old 09-22-2015, 03:32 PM   #3
bunce
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Hi - never had the "out of spec" error after ~100 miseq runs. As cheezemeister mentioned the only time we have had focus issues is due to under clustering - when we were dialing-in our quant. We also had a couple of runs 'stop' if they tip off the end of the amplicon (this also causes a focus issue) - the solution here is not to include cycles that you don't really need.
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Old 09-23-2015, 03:52 AM   #4
GenoMax
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@thermophile: In addition to the clustering issues mentioned above, bad libraries can also result in the instrument not finding focus. Out of curiosity were you able to successfully sequence a library that had encountered this error on a subsequent run without any changes?
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Old 09-23-2015, 05:17 AM   #5
thermophile
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All of our focusing issues are well into the run, which doesn't make sense. The majority have failed in R2. Looking at the thumbnails, the tiles seem pretty well focused then suddenly they'll stop. All of the failed libraries have eventually being successfully run though some have taken a few tries.

cheezemeister-The few times we've tried v3 600 cycle kits, we saw more dramatic drop in q score after ~200 & 400 cycles than what we see on v2 500. So it's not like the q score drop in the 600 kits is just a continuation of what you see in 500 cycle kits, it's actually worse.
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Old 09-23-2015, 05:36 AM   #6
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Quote:
Originally Posted by thermophile View Post
All of our focusing issues are well into the run, which doesn't make sense. The majority have failed in R2. Looking at the thumbnails, the tiles seem pretty well focused then suddenly they'll stop. All of the failed libraries have eventually being successfully run though some have taken a few tries.
Did that involve changing loading concentration/adding a phiX spike. I am wondering if there is a problem with your specific instrument. Generally getting your FAS involved will help move the case in the right direction.

Quote:
cheezemeister-The few times we've tried v3 600 cycle kits, we saw more dramatic drop in q score after ~200 & 400 cycles than what we see on v2 500. So it's not like the q score drop in the 600 kits is just a continuation of what you see in 500 cycle kits, it's actually worse.
This is a known issue with 600 cycle kits. Here is the thread: http://seqanswers.com/forums/showthread.php?t=59558
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Old 09-23-2015, 06:25 AM   #7
thermophile
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Geno, I really think there is something wrong with our specific instrument. our FAS and FAE have been here so much that I feel like we're old buddies. Old buddies who only see each other when we're annoyed with each other. My FAS has said that our focus issues seem to happen a little more often than most other instruments but that's as far as he's been willing to go. Hence my polling the interwebs to see if we really are experiences somehting unusual.

Sometimes we do increase the phix or add another genome to the reruns but not always.

eta, we always run at least 1% phix, up to 20% if we have no genomes to increase complexity of amplicon runs

Last edited by thermophile; 09-23-2015 at 09:27 AM.
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