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To remove the least reliable data from the analysis, the raw data can be filtered to remove any clusters that have “too much” intensity corresponding to bases other than the called base. By default, the purity of the signal from each cluster is examined over the first 25 cycles and calculated as Chastity = Highest_Intensity / (Highest_Intensity + Next_Highest_Intensity) for each cycle. The new default filtering implemented at the base calling stage allows at most one cycle that is less than the Chastity threshold.
The higher the value, the better. This value is very dependent on cluster density, since the major cause of an impure signal in the early cycles is the presence of another cluster within a few micrometers.
Very few clusters passing filter
Poor flow cell, perhaps unblocked DNA
Out of focus
A fluidics or sequencing failure
Bubbles in individual tiles
Too many clusters
High phasing or prephasing
Some of the causes may be at a single cycle. If the problem is isolated to these early cycles, it is possible that this filtering throws away very good data.
Base calling errors may be limited to affected cycles, and, as early cycles are fairly resistant to minor focus and fluidics problems, even the number of errors may be few. The filtering can always be set manually to some other values.
Check before assuming all the data are poor.
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