MiSeq intermittent blurry images.

pmiguel · 04-26-2012, 04:39 AM

Here is a quality plot of our ongoing MiSeq validation run:

Overall pretty good. But, if you look at one of the more poorly performing tiles you see:

Seems largely to be a tile-specific issue:

Any ideas what is causing this? Vibration seems unlikely because I would expect intermittent vibrations to effect the tiles randomly.

--
Phillip

ECO · 04-26-2012, 07:13 AM

I would guess that your stage tilt is out of spec or very close. Pretty quick fix for an FSE and tech support will replace the reagents. We had this problem on a couple of ours but after a secondary checkup they've been fine.

Whenever we see "swirly" images like this, that was the cause.

pmiguel · 04-26-2012, 10:04 AM

Quote:

Originally Posted by ECO

I would guess that your stage tilt is out of spec or very close. Pretty quick fix for an FSE and tech support will replace the reagents. We had this problem on a couple of ours but after a secondary checkup they've been fine.

Whenever we see "swirly" images like this, that was the cause.

Thanks. Actually the FAS did the install. I'll mention this to her. (It was just the phiX validation run, so we are not out any reagents.)

Phillip

ECO · 04-26-2012, 04:08 PM

I know...same with ours...the cluster density and other metrics may still pass their specs even with some of the image out of focus. The FAS/FSE had to come back and readjust the stage post install.

pmiguel · 05-04-2012, 12:22 PM

Quote:

Originally Posted by ECO

I would guess that your stage tilt is out of spec or very close. Pretty quick fix for an FSE and tech support will replace the reagents. We had this problem on a couple of ours but after a secondary checkup they've been fine.

Whenever we see "swirly" images like this, that was the cause.

Wow, you are good. FAS ran a check and stage tilt failed! It had passed during the install, but somehow got out of alignment. Should be fixed now.

We did a 2x25 nt run a couple of days back with the stage tilt presumably out of alignment -- worked fine. Could see no trace of this issue. Probably not that far off spec. Spec is -400 to 400 and the tilt was in the 700s when checked yesterday before it was re-aligned.

Actually my FAS wants to contact your FAS/FSE to find out if there is someway to lock that alignment down. But maybe it just happens sometimes during instrument "burn in"...

--
Phillip

ECO · 05-06-2012, 09:23 AM

Quote:

Originally Posted by pmiguel

Actually my FAS wants to contact your FAS/FSE to find out if there is someway to lock that alignment down. But maybe it just happens sometimes during instrument "burn in"...

Marc Rosoff is our guy.

Seems to be starting to happen on the second of our newest instruments as well...I'm hoping something is just loose from initial assembly and not something that will continually go out of spec.

Saddest part is that even when our runs start to drop to <70% cluster PF because of this problem, the instrument still produces enough in-spec data that ILMN won't replace the reagents.

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04-26-2012, 04:39 AM	#1
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 1,697	MiSeq intermittent blurry images. Here is a quality plot of our ongoing MiSeq validation run: Overall pretty good. But, if you look at one of the more poorly performing tiles you see: Seems largely to be a tile-specific issue: Any ideas what is causing this? Vibration seems unlikely because I would expect intermittent vibrations to effect the tiles randomly. -- Phillip

04-26-2012, 07:13 AM	#2
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,248	I would guess that your stage tilt is out of spec or very close. Pretty quick fix for an FSE and tech support will replace the reagents. We had this problem on a couple of ours but after a secondary checkup they've been fine. Whenever we see "swirly" images like this, that was the cause.

04-26-2012, 04:08 PM	#4
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,248	I know...same with ours...the cluster density and other metrics may still pass their specs even with some of the image out of focus. The FAS/FSE had to come back and readjust the stage post install.