When folks hear about my gig here at Adaptive, their first questions are always about the sequencing — and indeed we have some wicked cool machines for this in the lab. But our special sauce really is what comes before, and after, the Illuminas do their thing.
The “before” magic comes in the form of chemicals — our proprietary “primer mix” that drives the PCR or “Polymerase Chain Reaction” step. PCR is a pretty amazing process that won a dude the Nobel Prize back in 1993 and drives everything from criminal forensics to the immunosequencing we do here.
For our purposes, in order to get a good representation of the T- or B-Cells in your repertoire, we need to “amplify” the samples we receive (blood, tissue, etc.) pretty dramatically. Remember, your immune system is incredibly diverse, and many of the clones may only appear a couple of times in the sample — like just a few actual cells. We need to multiply the heck out of these guys to get enough that we can detect them during sequencing. We use PCR to do that.
The process works pretty much exactly like the old Faberge commercial that very few of you are old enough to remember but you can watch on YouTube. You start with one strand, and turn that into two, then four, then eight, and so on. A typical 20-cycle run of PCR will turn one strand into just over a million. Each cycle works like this:
- Mix your sample with an enzyme like Taq polymerase, a bunch of free-floating nucleotides and a bunch of “primer” DNA fragments. The primer matches a constant section of DNA you know is present on your sample, and kickstarts the DNA assembly process much like a seed crystal starts the awesome rock candy creation process.
- The enzyme induces the primer to attach at the right place to your sample DNA strand, and then adds matching nucleotides one by one from there along the sample. Once you’ve let that jiggle around for awhile, each strand will have been fully copied and bound to its mate.
- Now heat up the mix to about 95 degrees Celsius. This “denatures” the DNA strands, breaking the hydrogen bonds between them so they separate into individual strands again.
- Cool things down and start over with a new cycle.
So again pulling this back to Adaptive — remember from my last post that our task is to identify the key receptor sequences for T- and B-Cells that are created by randomly combining alleles from specific parts of your genome (“V” and “J” at the ends). While there are a bunch of possible alleles in play (and mutations that can happen too), we’ve been able to create primer libraries that capture the necessary sequences to hit pretty much the full complement. Those primers are a key part of our magic.
But of course, it’s never quite that simple. Our process is called “Multiplex PCR” because it uses a bunch of primers simultaneously — we have to do this in order to capture all the variants in your repertoire. But as it turns out, different primers can replicate at different rates. Oh crap.
This matters because we’re not measuring your original cells, we’re measuring a set that has been multiplied many times over. In order to be useful, the ratio of each sequence to the total has to stay constant — that is, if 10% of the cells in the amplified set are sequence X, then we need to be able to trust that 10% of the cells in the original set were too. But if things are multiplying at different rates, we’re obviously, well, screwed.
Happily, there is more Adaptive magic to the rescue. We’ve designed ways to track and measure this “amplification bias” for our primers and correct it with a combination of chemistry and computational tools. This is where the software starts to get pretty awesome too.
But it’s also where I’m going to quit for the day. Hope you have a great weekend!