In the wake of my previous blog post, I have received numerous bits of advice on how to make my set of COVID-19 candidate primers better. Today I am releasing my second version of the candidate primers. Here is a summary of the updates:

  • All data resources are being updated on a Git repository on BitBucket, allowing easy sharing among bioinformaticists. Feel free to fork & send me pull requests to make this resources better.

  • Latest spreadsheet report (version 2) with recommended candidate primers highlighted in green (marginal primers highlighted in yellow).

  • Added many additional columns to the report to help in candidate primer probe set selection, including %GC of the oligos, the melting temperature of the oligos, and amplicon %GC and length (relative to the reference RefSeq COVID-19 complete genome). Column ‘U’ in particular reports the difference between the probe melting temperature, and the highest melting temperature of the forward and reverse primers. You want this difference to be larger than 6 degrees Celsius, to allow for efficient probe hybridization. I sort the report first on the F1-score (column ‘M’, descending) and the probe-primers melting temperature difference (column ‘U’, descending), to make it easy to find the best primer-probe sets.

  • Documentation on the meaning of the columns in the spreadsheet report.

  • Added the Primer3 parameter file, so that everyone can see the choice of parameter settings used for generating these primers. Of course there’s no one-size-fits-all, as different kits will be run on different machines, and thus there will be a need to modify the parameters to fit the given protocol. I welcome feedback on how I can create additional parameter files for various protocols.

  • A FASTA file of COVID-19 primer-probe sets that are currenlty deployed in testing kits.

  • Fixing of the NCBI Blast database sequence identifier issue (see original post)

  • Improvements to the amplicon-calling code and other fixes to improve performance

I hope this is another step towards making this work more useful and accessible. Please let me know if you have any technical questions or bug reports (code bugs or documentation bugs) by creating an issue in the Issue Tracker. For all else, you can find me on Twitter or email me (see bottom of page).