is calculated using Unafold .
2Low complexity regions (repetitive sequence) are excluded from consideration as primers by setting a minimum entropy threshold for a primer candidate. The entropy of a sequence was computed by counting the numbers of occurrences of of the 64 possible trimers in the probe sequence, and dividing by the total number of trimers, yielding the corresponding frequencies . The entropy is then given by the sum of where the sum is over the trimers t with .
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