Tuesday, February 20, 2024

AGBT Follow-up: Ultima Genomics UG100, Volta Labs Callisto, N6Tec iconPCR

A confusion of ideas for AGBT follow-up have collided with the inevitable post-AGBT return-to-ordinary-life requirements.  To try to avoid a huge project that never gets completed, I'm breaking these up into multiple pieces.  First off, a look at reaction to the three big pieces I wrote before the conference or early during the conference: Ultima Genomics, Volta Labs Callisto and N6Tec iconPCR.  My comments are based on further thoughts on my part, discussions with other AGBT attendees and feedback I've gotten via social media, blog comments and emails/DMs.  Please keep it coming!  One of the great values of writing this is getting feedback - it illuminates questions I haven't considered and highlights gaps in my thinking. 

Ultima Genomics UG100

The Ultima Gold Sponsor lunch appeared to elicit both positive responses and certain areas of skepticism

I think the greatest positive response has been to ppmSeq, the ultra-accurate mode offered by Ultima.  The advantage of Q60 reads for needle-in-a-haystack problems such as detecting oncomutations in cell-free DNA is easy to grasp.  

A major area of skepticism is over Ultima's accuracy claims.  In particular, the question often raised is to what degree is Ultima putting their thumb on the scale by censoring difficult regions of the genome from the analysis, and is this any worse than what other vendors censor?  Clearly there needs to be independent comparisons of the variant calling; an opportunity for enterprising students given how much Genome-In-A-Bottle (GIAB) data is freely available for multiple platforms.

The question of homopolymer calling specifically also arose. One question I have as someone who doesn't do clinical variant calling on human (or to be honest, any variant calling on human genomes) is how many homopolymers do people really care about?  A clinical geneticist I met did mention a known pathogenic homopolymer variant in the cystic fibrosis locus.  Again, a great student project: cross-reference the variant calling on GIAB data with the ClinVar database of clinically-relevant variants and highlight the ClinVar variants with the most troublesome Ultima calls

The sheer size of the twinned instruments, emulsion PCR execution and sequencer, was noted by some - one person mentioned to me that their lab couldn't physically accomodate the pair.  

Mechanical reliabilty was another area of concern; at the Napa event both the company and several early access users had discussed early issues and the great improvements that had arrived with various "hardening" upgrades. I was asked in one private message whether any early access sites had thrown in the towel after reliability concerns, and when I brought this up with Ultima they said, no, none had.  But risk-tolerant early access users are very different from users wanting high uptime, particularly in a defined turnaround time testing product, so this will be an area to watch.

Volta Labs Callisto

I believe it is fair to say that Volta Labs' Callisto instrument intrigued many; electrowetting chemistry is fascinating to contemplate.  Most concerns were around an untested company launching a complex product with multiple consumables; it is certainly critical for Volta to launch on time in Q2 and launch a product line that delivers.  

The marriage of conventional liquid handling and electrowetting was flagged by some as an area of concern; each can have reliability issues.  

An area of skepticism is whether Volta has arrived too late since conventional liquid handlers are increasingly addressing the long read DNA extraction and library prep world - PacBio announced new protocols for Hamilton robots at AGBT and promises these will flow to other PacBio automation partners in the near future.  If side-by-side comparisons show a clear advantage to Volta in terms of insert sizes - and that will probably matter more on Oxford Nanopore which is in Volta's second tranche of supported protocols - then Volta wins; if not they may have harder sledding.

The >$100K price was also cited as a potential issue; at that price there are competing conventional liquid handlers that are more mature technology.  Of course, there's also OpenTrons at much lower price points, though OpenTrons has a very mixed reputation for reliability - asking about the perceived value of OpenTrons is a strongly polarizing topic.

Finally, I didn't detect much enthusiasm for the short read applications - but perhaps that's my own enthusiasm for the long read applications coloring things.  Automating hybrid capture steps may be of the greatest interest in this space, though if Element's on-flowcell capture scheme leads to imitation by other short read platforms it could steal that fire.

N6Tec iconPCR

I think I mostly sold people on the value of the N6Tec real-time thermocycler with complete control over each well's thermal profile.  The value proposition isn't hard to explain and easily resonates.  Whether the cost savings for NGS library prep will be as good as the company claims hit some skepticism.  Sometimes I saw a bit of skepticism also on the value of avoiding overamplification; I'm certainly sold on that aspect having been burned by it multiple times in my career.

The $100K pricetag - which the company admitted is at the high end of the range for a qPCR thermocycler - will probably inhibit adoption.  One public comment bemoaned how this will be prohibitive for many small labs that could make great use of the instrument.  I never asked the company if they plan on offering a leasing plan.

I'm not the only one wishing for a 384-well option; the value of 384-wells in high throughput environments is undeniable.

[2024-02-21 - fixed VoltaLabs instrument name to Callisto (not Calypso -- that's a serious mental error on my part & a failure to check]


SB said...

Thank you for the follow-up article. I am wondering why the N6Tec iconPCR instrument is not capable of performing real-time qPCR. I thought its capability to avoid over amplification combined with qPCR would be great to reduce PCR related variability in quantitative applications.

Keith Robison said...

SB: iconPCR is indeed a real-time qPCR instrument; that is indeed how it enables stopping amplification at a defined point in the amplification curve.

So yes, what you want is what it delivers and that is a key argument for using it: avoiding over-amplification and the consequent distortion of quantitative information in a sequencing pool.

If you could point where I've confused you, I'll try to fix it

SB said...

Keith: Thanks for the clarification. I went back to your article on this instrument to figure out why I seem to remember there is no qPCR capability, and found this sentence "It’s also true that if you wish to vary cycle times, the real time option isn’t available - at least for now". May be I misremembered what I read.

There was no confusion about what the instrument does - stopping amplification at a defined point; I was looking for how this is achieved. That I feel would help minimize PCR related errors in the area of analytical quantification that uses real-time qPCR. May be at some point there will be more details about the instrument that tells how things work.

Chris Streck said...

Hi SB,
I can confirm the system is a real-time qPCR machine and you're spot on with the intended use. The real-time monitoring and individually controlled wells were designed to reduce the PCR variability/artifact/over and under-amplification of NGS libraries. Happy to share more details as needed.

Keith Robison said...

SB: Ahh, that's an important detail that can be confusing

If you are only varying the temperature -- what temperature in the cycle, then the qPCR option is available and you can program the thermocycler to stop wells when they hit a stage in the cycle

If you vary the cycle times between wells, the qPCR option is not available. I haven't asked careful enough questions to understand why not; it may be enabled with future software versions but perhaps not.

Does that clear it up?

Joe said...

"Sometimes I saw a bit of skepticism also on the value of avoiding overamplification; I'm certainly sold on that aspect having been burned by it multiple times in my career."

So what kinds of problems do you see from overamplification? Aside from the just greater difficulty QC'ing and quantifying the library.

Keith Robison said...

You'll find more in the original post on iconPCR, but some of the sins of overamplification
1. Quantitation / diversity is muddled - N6 presented data on this for rRNA amplification
2. Chimaeras are more likely to form during overamplification - also in the rRNA data & I've seen this in other datasets
3. Quite likely uniformity of coverage suffers; the more amplification, the more effects of preferential amplification will be seen