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Old 11-04-2009, 07:16 AM   #14
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Location: France

Join Date: Nov 2009
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Originally Posted by pmiguel View Post
But I think there is a second, related, issue in play. About this, Roche is more typically inscrutable, but one hears whispers. That is, one is led to believe that the image analysis software is sensitive to well-to-well bleed-over. Two beads sitting in adjacent wells of a PTP, if they emit too similar a sequence, are presumed to be "overlapping" and one or both are discarded. I'm not privy to what the threshold is for such a filter, if it does even exist, to kick in. It is of some significance, however; because SMRT cap adaptors, for instance, if they are sufficient to trigger high levels of bleed-over filtration, would be problematic. Especially if, as I believe is the case, the fragmentation broken ends are less often ligated than the extant capped ends.

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 ?
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