Validate PCR DNA primers against whole de novel genome

Question

[qwzhang0601]

I want to test gene copy number of some genes. I designed the primer using primer3 and what to test whether the primers are specific to my genes. I know some tools can do this. For example, I tried primer-blast, but it can not work when I test it against the whole genome (about 3G base pairs). I also know others (e.g., UCSC In-Silico PCR), but since our genome is new, I can not provide my genome. Do anyone knows some tools can handle large genome to check specificity of primers?

Thanks

Answer 1

[trausch]

Dicey can do this. You first need to build an FM-Index and the standard FASTA index of your genome

dicey index -o new_genome.fa.fm9 new_genome.fa.gz

samtools faidx new_genome.fa.gz

Then you can search primer pairs across the entire genome.

echo -e ">Primer_forward\nGCCCCATAGGTTTTGAACTCA\n>Primer_reverse\nTGATTTGTCTGTAATTGCCAGC" > primers.fa

dicey search -c 45 -g new_genome.fa.gz primers.fa

The default output is a JSON file that you can convert to text using the provided script (scripts/json2txt.py). Dicey is available as a static binary or on Bioconda.

[qwzhang0601]

Many thanks. Is there a publication and manual for dicey? I am looking at MFEprimer-3.0 (https://www.mfeprimer.com/), have you tried this before?

Thank you

[trausch]

No publication yet, there is only the GitHub README. But I am happy to help if you encounter problems with dicey. Just open an issue in the repo.

[qwzhang0601]

Ok. Thank you very much.