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Old 04-04-2013, 08:51 AM   #10
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Quote:
Originally Posted by GenoMax View Post
The difference was not as dramatic as your plots but the top surface was better. We have several HiSeq's and I have not heard of recurring bubble issues.
I can only speculate, but if this is a common issue, then I don't think it would be noticed for the most part. You actually need to make the link between "some high error tiles in the bottom surface of my flowcell" and "transient bubbles in my flowcell".
Because they are transient and not that common, there is just a slight degradation in the data quality from reads from the bottom surface. These end up being "N's" or very low quality bases in the final sequence.
Quote:
Originally Posted by GenoMax View Post
You may want to ask your friendly illumina engineer to look at the possibility of a small leak somewhere, specially if you see the bubbles regularly.
I do, but there are a certain number of bubbles you see in flow cell lanes during, for example, a pump check. They actually train you to see these small bubbles coming through as a method to ensure there is flow.

To me that says some bubbles are "situation normal". Given that, then you expect a number of bubbles in the scan buffer as well.

At the risk of testing you patience on this, the error plot I showed previously:



If you right-click on that pane in SAV and choose "Autoscale" you get a full scale assessment of the bad tiles:



So maybe it isn't the tiles that have 4% error rates that have the bubbles, but the tiles that have higher ones? The flowcell pane of SAV appears to cap the error rate color scale at 4% unless you manually adjust it higher. I have to hover the cursor over the scale and use my scroll wheel to do that. Then I can focus on just the tiles that have pretty high (>4%) error rates:



Then you can click and drag on the color scale to get to a place in their palate that really highlights the high error tiles:



So I can see nine high error tiles:


For each the cause would be the bubble that is clearly visible.
You don't see that at all?
How about anyone else?
If not, then my instrument has an issue, that could presumably be fixed.

I guess on the scale of things, the issue is minor. It only effects the clusters covered for that one cycle. 9 of 384 tiles or 2.3%. Even then the tiles only suffer a few percent loss in data. So maybe I am being too "glass half empty here"?
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Phillip
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