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I could not find any bacterial species in the drop-down list for genomes in https://genome.ucsc.edu/cgi-bin/hgPcr

Any kind of help would be appreciated. Other links for in silico PCR did not really work or perhaps I do not know the procedure well. Hopefully this query aids others in the future seeking a similar solution.

Edit: I wanted to check the primer pair 27F and 1492R which are part of Oxford Nanopore 16S library prep kit (SQK-16S024) on a bacterial genome.

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  • $\begingroup$ Your question lacks clarity and detail : what is the purpose of the PCR ? What would you want to do with the amplicons ? $\endgroup$
    – CaroZ
    Commented Jul 6 at 10:18
  • $\begingroup$ Need to check why the Oxford Nanopore 16S024 kit's primer pair (27F and 1492R) is resulting in two bands instead of a single band of 16S rRNA gene. Once I get the amplicons, there's library prep followed by sequencing on Nanopore to understand which bacteria were present in the samples of interest. $\endgroup$
    – Rahul Dhargalkar
    Commented Jul 8 at 5:49
  • $\begingroup$ What size bands? This primer set has been found to amplify some 18s eukaryotic rRNA genes, especially when using a lower annealing temp that is more permissive to minor mismatches. TestPrime is a good in silico PCR tool, but it only runs on the Silva SSU and LSU rRNA databases (not whole genomes). arb-silva.de/search/testprime $\endgroup$
    – MikeyC
    Commented Jul 8 at 15:03
  • $\begingroup$ @MikeyC The size of the band should be around 1500 bp since these primers are for full-length V1-V9 16S region. And you are correct, I did come across literature which talk about cross-reactivity of 16S primers with eukaryotic 18S genes. I was not aware of this until now so thank you very much! I shall check out your suggestions and if you want to know, I could keep you posted, formally. $\endgroup$
    – Rahul Dhargalkar
    Commented Jul 10 at 5:00

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I would suggest using NCBI's Primer-BLAST tool. Formerly I would have recommended their e-PCR tool, but it has been retired. This retirement link has a lot of instructional information for how to use the tool.

You can upload your own assembly/accession, or very likely most well known species of bacteria will be in their database or in refseq.

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  • $\begingroup$ Thank you for the response. I shall try to learn how to effectively use the Primer-BLAST. I tried it but could not follow well. In the meantime, could you give more pointers? I am supposed to upload a reference genome as template, right? I tried that with the primers but it did not work. $\endgroup$
    – Rahul Dhargalkar
    Commented Jul 8 at 5:52
  • $\begingroup$ @RahulDhargalkar You could upload a reference genome, or you could select one of the included genomes that is in RefSeq. I'd suggest following the instructions here: ncbiinsights.ncbi.nlm.nih.gov/2017/06/28/…. I can try to add a working example later when I have more time. $\endgroup$
    – Maximilian Press
    Commented Jul 8 at 16:24

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