Advances in Bioinformatics / 2014 / Article / Tab 2

Research Article

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

Table 2

Parameters used for primer design in in silico examples and MHV example presented here.

In silico primer settingsMHV primer settings

Primer length range18–2518–27
range allowed160–65°C58–65°C
Number degenerate bases allowed per primer53
Minimum distance of degenerate base to 3′ end of primer3 nt 3 nt
Minimum trimer entropy allowed (to avoid repetitive sequence)23.53.3
Maximum length of homopolymer allowed4 nt5 nt
GC% range allowed20–8020–80
Minimum primer dimer −6 kcal/mol−15 kcal/mol
Minimum hairpin −5 kcal/mol−12 kcal/mol
Primer selection iterations13

is calculated using Unafold [6].
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 .