Tuesday, February 06, 2024

iconPCR: Super-Flexible qPCR Thermocycler Oft Dreamed, Now Delivered

Has there ever been a product you’ve just wanted to have, but it doesn’t exist? That keeps popping up in discussions - “if only we had X this project would go so much faster!”. Well, N6 Tec’s automation-friendly $99K i96 well iconPCR thermocycler is that to me. Launching at AGBT, it’s the gadget I’ve wanted repeatedly at Codon Devices, Warp Drive Bio and now Ginkgo Bioworks. It won’t solve all your PCR challenges, but it certainly gives new options to customize PCR like never before. And for many NGS labs, it offers major streamlining of PCR-based library construction protocols while also delivering superior data. How? By being a thermocycler where every well can run its own thermal profile and each well can go dormant once a desired level of amplification is achieved 

Overamplification is not your friend; indeed it is often your enemy. In an ideal world, any PCR reaction would be amplified just enough and no more. It’s the late cycles that are most prone to chimaera formation when amplifying closely related templates for 16S or protein engineering or such. If you wish to preserve quantitation, and diversity, amplify as little as possible! Or if you wish to have as close to uniform coverage across a genome. But on a conventional thermocycler, every well must undergo the same number of cycles. There are protocols where qPCR is used and the amplification halted when some criterion is reached, but that criterion isn’t individualized to each well - you can stop early, but perhaps too early for some wells and not early enough for others. And you must watch your thermocycler; not the best use of a skilled scientist's time. 

Of course if you could have each well halt cycling at the same quantity of amplification product, you now have a normalized set of samples ready-to-pool without any individualized aliquoting or dilution. It also obviates trying to normalize the inputs upfront by quantifying them and diluting them to a standard.  And since the wells will be ready-to-pool, pooling can be upstream of bead cleanup to remove primer diners and unincorporated primers - another simplification of the workflow.  

So in the NGS space iconPCR offers better preservation of the diversity of the input material, fewer chimaera artifacts, simpler workflow and inherent normalization which allows better flowcell packing and fewer library dropouts.   And all that goes double for precious samples where you can’t afford to make mistakes — ancient DNA or FFPE. 

Now imagine applying this in other areas.  For examples, the ARTIC protocol for SARS-CoV-2 (and other virus) sequencing works great on high viral load samples, but can encounter problems with amplicon dropout on low viral load samples.  Even more so, it is often recommended not to run the protocol with plates containing a mixture of very high viral load samples and very dilute viral load - you often get huge numbers of reads from the high viral load and dropouts fro the low.  And viral sequencing often places a premium on getting results turned out quickly - simpler protocols definitely win here.  I’d love to see iconPCR applied to ARTIC on a collection of samples of widely ranging viral load; the autonormalization would certainly be useful and I’m wondering if some of the amplicon balance problems seen on high viral load samples could be tamped down with fewer amplification cycles.  And just imagine testing every number of cycles from 8 to 32 on a plate of a standard sample to explore this?  With conventional thermocycler, that’s going to be a lot of PCR runs to set up - with iconPCR you could run 4 samples in parallel on a single run.

Back at Warp Drive Bio we had two eras of degenerate PCR primers for trying to fish out a few  key genes diagnostic of our favorite class of biosynthetic gene clusters. For most endpoint PCR I had a standard protocol - suggested by the best cloned I know - which is to set the design melting temp to 70-72 degrees — hot is best!  Of course, we were primarily working with 72% GC organisms so that was another reason to run hot.  But degenerate primers aren’t ordinary primers and the pool doesn’t have a single melting temperature - plus you want the core of the primer as short as possible to minimize the number of degenerate positions or any. One primer will be present in vanishingly small quantities, Tuning a degererate PCR protocol is challenging — but what if we could explore dozens of different thermal profiles in a single run?

So think of all your challenging PCRs - the PCR assay development that went many rounds of exploring thermal conditions.  Gene synthesis PCR that didn’t quite go as planned or had the desired component as a minor fraction of the mixture — we had plenty of those at Codon. Amplifying long templates in a mutagenesis campaign or to generate ultra-low input libraries for PacBio. 

Or, as below, in a 16S rRNA profiling experiment - measuring diversity is your goal. In that cases, which of the diversity plots believe - each column a different environmental sample - would you prefer?

Gradient PCR instruments have existed for a long time, so it wasn’t the case that you were stuck with the same temperature profile in every well. But having a crude gradient across the plate is not the same as having precise control over each well.

In terms of operation, N6 has thought about what the market wants.  They say it can accept any standard PCR 96 well plates or strip tubes. N6 isn’t in the reagents buisiness; just use your favorite mastermixes.  It’s designed with automation in mind, with the plates introduced via a drawer that a gripper robot can access (shown open in the photo below).

When breathlessly extolling my enthusiasm for iconPCR at AGBT, several people asked a very good question: if desire for this instrument was always so great, why are we seeing it only now? And why from a company nobody knew anything about until around the product launch at AGBT?

Talking to the company - small but stocked with individuals who have put in many hours at major thermocycler manufacturers such as BioRad - one bit of enablement was electronic components which were originally developed for electric vehicles. I think on the company standpoint, it's too easy for the big players to stick to a very conservative product plan - this is a radical departure. And the pandemic played a slowing role - N6 Tec had just gotten started (quite literally in a founder's garage) and then some of the key components became difficult to source or would have the price go up even after they ordered. At times, the company stockpiled electronics they hadn't yet committed to, fearful that the alternative was for development to stall when it was realized the component was needed but then couldn't be obtained.

Okay. What’s the catch?  Well, it is a new instrument and N6 Tec will have to demonstrate they can deliver the instruments and that they are reliable in the field. It’s only a 96 well instrument - yeah, I’m always greedy for Moore. And only one optical channel; again with the greed - I’m the guy when presented with paradise would say”I have notes”.  N6 says both capabilities are on their product development roadmap, but no timing yet. It’s also true that if you wish to vary cycle times, the real time option isn’t available - at least for now.

But that's mostly minor quibbles - if N6 Tec's iconPCR instrument is reliable, it's going to find a home in many labs. Some crafty consumers will buy more than one instrument.

1 comment:

Dale Yuzuki said...

Great to see this kind of advance in something as mundane as PCR. Lots of potential for sure - next up is ability to execute (on N6 Tec's part). Many thanks for this update Keith - and say 'hi' to all my friends at AGBT24! (Alas could not wrangle attending this year...)