In the same gel, a DNA ladder (Invitrogen, Carlsbad, CA, USA) containing 100 2000 base pairs was added.
Furthermore, to allow for multiplexing, a sample-specific barcode (one to three base pairs) was added to the design between the adapter and the annealing primer.
Because nucleotides are not allowed in matched base pairs to base pair to any nucleotide other than its matched partner, helices that are pseudoknotted once the matched base pairs are added are allowed to form.
Inner and outer base pairs are added as long as the average reliability of the inner or outer extended stem, respectively, is larger than the threshold δ, and the probability of the partial structure is greater than or equal to γ either in the evolutionary or the thermodynamic model.
For each base pair in the WS170 build of the C. elegans genome, the reads mapping to the plus and minus strand of that base pair were added together to give an overall score.
It is also possible that as a result of mutations more than one base pair are added during the repair and replication phases.
If both positions S i and S j of a base pair of the current solution (i, j ) ∈ P sol are constrained by C seq, this base pair is added to the target structure P C. In a final step, the length-normalized base pair distance d s t r between P sol and the compiled target structure P C is determined.
As an additional C−G or U−A base pair is added to the top of stem 2 to give PSK-CG and PSK-UA, SPM and NUPACK predict the free energy for the pseudoknot by adding an additional INN-HB nearest-neighbor stack (−3.3 and −2.4 kcal/mol, respectively).
For this procedure, no existing base pairs were removed, no pseudo-knots were allowed and exclusively Watson-Crick pairs were added (see fig. 5 for examples).
