If custom ratios are required, then for each mixed base position, the IUB code followed by a colon with a listing of the desired ratio are both included in parentheses. The ratio must be written in order of A, C, G, T (or A, C, G, U for RNA). The first
instance of the hand-mixed base must name and define the ratio, all subsequent identical insertions only need to include the name. For example, an N mixed base with 25% of each base would be written as: (N1:25252525). Each mixed base in the sequence
with this ratio can be notated with (N1). See below for what this looks like when incorporated in a sequence.
- 5′ ACT GTA CCG TAT TCC (N1:25252525)(N1)(N1) TTA (N1)(N1)(N1) ATA 3′
Up to four unique hand-mixed ratios can be included in an oligo sequence. Each of these ratios must also include a unique name. See below for two examples.
- Example 1: 5′ CAT (N1:25252525)(N1)T (N2:20202040)(N2)(N1) G(N2)A 3′
This oligo has three insertions of a 25% for each base mix (labeled N1) and three insertions of a 20% A, 20% C, 20% G, 40% T mix (labeled N2).
- Example 2: 5′ AGG (K1:00004060)(K1)(K1) (D1:10002070)(D1)A GTA 3′
This oligo has three insertions of a 40% G, 60% T mix (labeled K1) and two insertions of a 10% A, 20% G, 70% T mix (labeled D1).