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Old 04-03-2013, 06:20 AM   #1
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Question How to reduce flowcell bubbles in the HiSeq?

A maddening property of the HiSeq is that it does not seem to take any steps to eliminate bubbles from the liquids it passes through the flow cell. Does anyone have methods to reduce their numbers?

Generally this has no effect on the top surface tiles but bubbles in the scan buffer prevent clusters beneath them from being scanned. Worse, bubbles near the bottom ports of the flow cell can cause imaging issues for the entire swath! (This is called "bottom middle swath" or "BMS".)

Further, the number of bubbles seems highest in the first few cycles, the very worst time for them. Any clusters covered by a bubble during the first 4 cycles will not pass filter -- are effectively lost. BMS causes 1/6th of the clusters in a lane to be lost.

Just to be clear, I do not mean bubbles being pulled through the rims of the flow cell gaskets ports. I can see these during a pump test using a pair of +6 "reading" glasses I bought from Amazon and a flashlight (or "torch" as it is known in other "english" speaking countries). This is always caused by tiny fragments of dust laying across the rim of the effected gasket port. Again, keen eyesight or +6 glasses may be needed to visualize these.

I mean the bubbles that are either in the scan buffer or form during pumping. (I presume the pumps produce enough vacuum to cause a certain number of them to form by solution de-gassing.)

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