PriSeT: Efficient De Novo Primer Discovery

Marie Hoffmann; Michael T. Monaghan; Knut Reinert

There exists computer-assisted approaches in which a manageable subset of references of organisms that are expected to show up, is collected and serves as input to compute a multiple sequence alignment (MSA). Then a variation or entropy score is computed given the nucleotide distribution for each position of the alignment. Low entropy regions are then analyzed for serving as primer templates and regions with high inter-species entropy as barcodes (Hadziavdic et al., 2014) . MSA approaches require the input data set to be small, phylogenetically close, and curated in order to produce meaningful alignments. Needless to say that in metagenomics this approach is only chosen post experimentum when analysing organism groups that turned out to be difficult to detect with PCR. Online tools like Primer Blast/Primer3 3 (Ye et al., 2012) take off the burden to locally set up a software system by providing primer searches as on online service. Users provide as input a GenBank accession or a FASTA file. NCBI's Primer BLAST uses Primer3 to design primer sequences and then submits a BLAST search against a user-provided file. Surprisingly, Primer Blast does not process files with multiple references and is therefore inapplicable in a metabarcoding setup.