Orthogonal Oligo Design for Fluorescence In Situ Hybridization (OOD-FISH) is a pipeline for the identification of oligonucleotide sequences that do not hybridize to a reference genome of interest. Such orthogonal oligonucleotides can easily be used as barcodes for PCR or similar protocols.

OOD-FISH can be used to identify the largest set of oligonucleotides (from the input) that:

Please, note that OOD-FISH does not include any filters based on secondary-structures of the oligonucleotide. This can be easily achieved with available software, like OligoArrayAux.

The pipeline includes the following steps:

  1. Sequences (input) generation.
  2. Alignment against reference genome.
  3. Calculate self-dimerization free energy (SDFE) of every barcode candidate.
  4. Filter candidates based on maximum homology and SDFE.
  5. Calculate pair-wise hetero-dimerization free energy (HDFE) of selected candidates.
  6. Filter based on HDFE.
  7. Select largest barcode set with Parallel Maximum Clique (PMC) algorithm.