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Old 07-07-2010, 10:41 AM   #3
Location: Frederick MD, USA

Join Date: May 2008
Posts: 39

Another phenomenon at work here is what Illumina calls 'phasing'. Remember that each cluster contains ~1000 (supposedly) identical molecules, all of which are undergoing reverse-strand synthesis.

In a perfect world, the chemistry works perfectly, and at each cycle each molecule has one base+dye+blocker added, and has dye+blocker removed after imaging. In the real world, a small fraction of the molecules doesn't come out right: they miss a cycle, or get an extra (unblocked) base added.

The cumulative effect of phasing over many cycles means that eventually the cluster has so many molecules out of sync that there is no clear base call possible, and miscalls start to happen. Quality scores, which are derived from the ratios of the intensities start to degrade. Eventually, you have to stop.

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