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Question: Validate PCR DNA primers against whole de novel genome
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gravatar for qwzhang0601
23 months ago by
qwzhang0601 • 70
United States
qwzhang0601 • 70 wrote:

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
in sillico primers • 481 views
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modified 23 months ago by trausch • 1.6k • written 23 months ago by qwzhang0601 • 70
1
gravatar for trausch
23 months ago by
trausch • 1.6k
Germany
trausch • 1.6k wrote:

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.
ADD COMMENT • link modified 23 months ago • written 23 months ago by trausch • 1.6k

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

ADD REPLY • link written 23 months ago by qwzhang0601 • 70

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.

ADD REPLY • link written 23 months ago by trausch • 1.6k

Ok. Thank you very much.

ADD REPLY • link written 23 months ago by qwzhang0601 • 70
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