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Old 11-04-2009, 09:58 AM   #15
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Quote:
Originally Posted by jmbon View Post
Thank you Phillip, this was a quick and informative answer. I do not fully follow your point about primers and bleed-over, though. If a primer sequence cause bleed over from well-to-well, many PCR products should be difficult to sequence in depth, and presumably not only SMART RT-PCR products. In our run with Evrogen RT_PCR kit, reads bearing in 5' the M1 cDNA primer are indeed in large excess (73%, for =<50% expected), so at least the M1 sequence seems safe for that matter. Could it be that what is bleeding is rather the too bright light of the polyT strech that follow every other 5'M1, making the neighbor beads difficult to read ?
I did not mean that the adaptor sequence caused bleed-over, only that it might look sufficiently like bleed-over to trigger the bleed-over filter.

Again, this is nearly pure speculation on my part, but the the reads that trigger the bleed-over filter will not be seen among your filter pass reads. They would have been filtered out.

I presume, the bleed-over filter would only be triggered if two beads in close physical proximity gave a similar sequence for some number of flows.

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Phillip
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