Dear Agata, what is your target organism? One tool is better for bacteria, another one is better for humans.
these people describe step be step what to do, for mouse and humans.
Do you need primers for PCR or NGS? Somewhere I saw a list of NGS-primers, I can try to find it.
Here you can choose what exactly you need, there are a lot of choices:
This paper describes the tool for viruses and bacteria.
Below I cite the paper above:
“Several primer design programs are available (OLIGO , OSP [7, 8], Primer Master , PRIDE , Primer3 ). Regardless of each computational working strategy, all of these use a set of common criteria (e.g., G/C content, melting temperature, etc.) to evaluate the quality of primer candidates in a specific target region selected by the user”.
“Alternative programs are aimed at more specific purposes, such as selection of primers that bind to conserved genomic regions based on multiple sequence alignments [12, 13], primer design for selective amplification of protein-coding regions , oligonucleotide design for site-directed mutagenesis , and primer design for hybridization . Usually, the design of truly specific primers requires the information of the complete nucleotide sequence. This is the starting point for most of the programs described in the literature. However, the need of designing specific primers is not always accompanied by the complete knowledge of the target genome sequence”.
And this is a problem. “However, the need of designing specific primers is not always accompanied by the complete knowledge of the target genome sequence”.
Their conculusion is optimistic:
"In general, the results suggest that FAS-DPD could be used to design generalized degenerate primers for detection of known or unknown members of gene families or organism families, including different types of pathogens".