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Old 10-18-2017, 02:11 PM   #4
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Location: Denmark

Join Date: Oct 2017
Posts: 7

Have a look at your intensities of the first 25 cycles (e.g. in the sequence analysis viewer) (which might not only be a problem for amplicon sequencing). Below a quote from a Illumina white paper:

The %PF calculations involve the application of a chastity filter to
each cluster. Chastity is defined as the ratio of the brightest base
intensity divided by the sum of the brightest and second brightest
base intensities. Clusters “pass filter” if no more than 1 base call has
a chastity value below 0.6 in the first 25 cycles.
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