Up at 5AM: The 5AM Solutions Blog

Designing Assays: The Oligo Craftsman vs. The Primer Jockey

Posted on Thu, Dec 08, 2011 @ 06:00 AM

assaysI have been designing assays for some time now. Starting with a few oligos in graduate school, I kicked up my PCR primer design experience out of school when I went to work for a genome center. Then there was my first blunder--ordering a few hundred dollars worth of primers with the reverse primers in the wrong orientation. I broke the news to my boss, resignation letter in hand (it hadn’t occurred to me that we probably spent more than that in one week just on pizza). Later in my career, the stress level went up a few orders of magnitude when I had to design tens of thousands of probes for SNP genotyping panels. That’s when I developed my addiction to TUMS® and came to the realization that my reward for a job well done was invisibility. With assay design, people only look for you when something’s wrong.

Jockeys and Craftsmen

I’m not a molecular biologist nor a biochemist. I can conceptualize how different probe and primer chemistries work, but that is not my specialty. What I am good at it is taking assay design rules and guidelines and actualizing them as a software pipeline. People like me go by several titles: Base Boy, Nuc Nerds, the Oligarch of Oligos… but I prefer the moniker “Primer Jockey.”

As a Primer Jockey, I have met from time to time another assay design professional whom I call the “Oligo Craftsman.” With almost unimaginable patience, these folks take each assay target and hand-craft their oligos, base by base. Estimating melting temperatures by counting C’s and G’s and eye-balling hairpin structures, they bring years of knowledge about their assays--and perhaps a little black magic--to the process of design. Tragically, Jockeys and Craftsmen rarely see eye-to-eye--the Craftman complaining that the Jockey’s tools don’t “do what they want” and the Jockey left exasperated by the Craftsman’s unscalable and seemingly inconsistent methods.

Data Will Bring Us Together

Ultimately, I believe only data can bring Jockeys and Craftsmen together. The Craftsman needs complete transparency--in the form of data--from the Jockey on what assays were considered, which ones were not and why one assay was chosen over another. This allows the two sides to debug and iterate to a process that meets both sides’ expectations. Jockeys need data in the form of assay performance results. Does the data support higher failure rates when one of the Craftman’s “rules of thumb” is not met? Unfortunately, assay performance data rarely leaves an institution. Even worse, journal articles written on the subject of assay design frequently do not share raw performance results or oligo sequence, nor do they submit data to a resource like the Probe Database. Until this kind of data is readily available, the Jockey and Craftsman will likely remain at odds.

Our Challenge to You

Our motto at 5AM is “Think, Do Well, Be Good, Stand Up.” In that spirit, we’re looking forward to sharing some of our assay design tools and methodologies with the community in the months ahead--stay tuned. As an initial step to improving the general knowledge of primer and probe oligo design, however, we offer this carrot. One of the barriers for scientists to make their assay results and oligos available is the general headache of formatting their data into the required formats of resources like the Probe Database. For those without bioinformatic resources, this can mean hours of tedious and error prone Excel manipulation. With some programming ability, this can often go much quicker and that is our offer to you--If you have nucleic assay data of any kind that you’d like to submit to the Probe Database, we will help you do this for free when our resources allow. This is our way of trying to get as much data out there as possible to the benefit of all. Finally, if you’ve read a paper that has useful assay design data, please comment below. Thanks!

-J Ireland, 5AM Solutions

Tags: genomics tools, bioinformatics, assay design, assay design tools, oligo design, assay design methodologies, bioinformatic resources, probe database, nucleic assay data


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