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 settings	MHV primer settings
Primer length range	18–25	18–27
𝑇𝑚 range allowed^1	60–65°C	58–65°C
Number degenerate bases allowed per primer	5	3
Minimum distance of degenerate base to 3′ end of primer	3 nt	3 nt
Minimum trimer entropy allowed (to avoid repetitive sequence)^2	3.5	3.3
Maximum length of homopolymer allowed	4 nt	5 nt
GC% range allowed	20–80	20–80
Minimum primer dimer Δ𝐺	−6 kcal/mol	−15 kcal/mol
Minimum hairpin Δ𝐺	−5 kcal/mol	−12 kcal/mol
Primer selection iterations	1	3

¹𝑇_𝑚 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 𝑆_𝑖 of a sequence was computed by counting the numbers of occurrences of 𝑛_𝐴𝐴𝐴,𝑛_𝐴𝐴𝐶,…,𝑛_𝑇𝑇𝑇 of the 64 possible trimers in the probe sequence, and dividing by the total number of trimers, yielding the corresponding frequencies 𝑓_𝐴𝐴𝐴,…,𝑓_𝑇𝑇𝑇. The entropy is then given by the sum of −𝑓_𝑡log₂𝑓_𝑡 where the sum is over the trimers t with 𝑓_𝑡≠0.