PriSeT: Efficient De Novo Primer Discovery

Marie Hoffmann; Michael T. Monaghan; Knut Reinert

Each k-mer has to pass a chemical filter which checks molecular property constraints (see C s in Table 2 ) that have to hold for single primers independent of possible matching partners. Applying the filter directly after the frequency step helps to reduce the number of k-mers by orders of magnitudes before running the combine procedure. The constraints C s that are currently checked by PriSeT are amplicon length range, melting temperature, CG content, mono-or dinucleotide runs, and self-annealing patterns (see Table 2 ). amplicon ranges are constricted, because we first need to ensure that reads will overlap for the read merging step in a metabarcoding pipeline, and secondly, reads are usually trimmed to the same length -a precondition for most OTU clustering algorithms. The melting temperature in a PCR corresponds to the state where half of the DNA duplexes will be dissociated. Only when template DNA is single stranded, primer sequences can bind and the enzymatically driven elongation can take place. The temperature for the denaturation phase can be adjusted, but too high temperatures will lead to irreversible damage of the ingredients. Another criterion for primers being not too associative to the template is their CG content. Not only are C-G bonds twice as strong as A-T bonds, they are also more prone to mis-pairing. The recommended range is 40 -60 % in proportion to their length. Mono-or dinucleotide runs are patterns where a single nucleotide or dinucleotide occurs consecutively repeated, i.e. σ x or (σ 1 σ 2 ) x with σ ∈ Σ, σ 1 = σ 2 , and x ∈ N . These patterns tend to misprime. A maximal accepted number is four. Self-annealing (or self-dimerization) occurs when copies of a primer form stable dimers. If their binding energy is relatively high, a significant amount will rather bind to primers than to the template and thereby deteriorating template am-. CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.06.027961 doi: bioRxiv preprint plification. The free Gibb's energy is a measure for the bond strength. It numbers the energy that is released during bond formation and should not be below -6 Jmol −1 . The amount depends on the positions and types of nucleotides involved, and can only be determined precisely via experiments. When looking at an alignment of two oligomers or primers, as a rule of a thumb, there should be not more than four annealing nucleotides in a row (connected annealing pattern) or not more than 50 % of the sequence (disconnected annealing pattern) involved in the bonding as shown in Figure 1 .