Tagify: seqWell's Line of Tagmentation Reagents Awaits Your Creative Thoughts!

One of the most important enzymes in the sequencing world, one which enables spectacular creativity on the part of novel assay designers, is Tn5 transposase.  Personally, I spend many times each month thinking about how to use Tn5 and its ability to tagment - both tag and fragment - input DNA. There’s even reports that Tn5 can tagment RNA-DNA hybrids such as from reverse transcription or even long single-stranded DNA.  I’ve covered seqWell in the past,with their fully kitted reagents; now the company (which just turned ten) is launching a Tagify product line that is focused on enabling NGS dreamers to easily explore new Tn5-based library preparation methods.

mRNA Therapeutic / Vaccine Quality Control: A Major ONT Opportunity?

Oxford Nanopore is in the process of morphing into a product-focused company, and so must identify specific markets in which they believe nanopore sequencing can compete or even dominate.  One such market that was spotlighted this year at London Calling is the quality control of mRNA therapeutics, where nanopore sequencing may be able to replace a kitchen sink of technologies and often provide superior data.

Pharmaceutical and diagnostic quality control is both similar and very different to research.  While many sequencing research experiments are to some degree a fishing expedition, in a quality control assay very specific hypotheses are tested with specific, pre-determined thresholds.  Consistency of results is the most critical; an assay run today must be comparable with one run last month or last year.  These markets may be less sensitive than research to cost; if a QC test is part of qualifying a vaccine batch which will sell for millions of dollars, spending a thousand on that assay isn't unreasonable at all.

It's worth reviewing the process of how mRNA vaccine drug substance are made. The initial vaccine design is synthesized into a plasmid; this design includes a poly-A tail followed by a restriction site (which cannot occur within the vaccine design, though it could occur elsewhere in the promoter backbone).  Enormous batches of plasmid are grown in E.coli and extracted and then linearized with the restriction enzyme that cuts after the poly-A tail and has no sites .  In vitro transcription is used to transcribe the linear template, with the nucleotide mix containing a uridine analog such as 5-pseudouridine in place of uridine.  If the BioNTech process is used, then the nucleotide pool also contains a guanine analog which contains a 5' cap structure (CleanCap).  If Moderna's process, then the in vitro transcription product is treated with a capping enzyme (typically Vaccinia Capping Enzyme aka VCE; please see conflict-of-interest disclosure at the bottom of this piece). After purification and concentration of the active drug substance (removing nucleotides, process enzymes, uncapped product, etc), drug product is ready for the finish-and-fill steps of encapsulating it in the lipid nanoparticles and filling vials for distribution.

QC is all about detecting what might go wrong and ensuring consistency of product.  mRNA therapeutics and vaccines are complex products, with many possible parameters to measure.

First, there's the question of "is this the right product?".  mRNA vaccines continue to evolve and expand in scope, with new designs targeting specific SARS-CoV-2 variants, influenza and RSV vaccines.  If a vaccine product should be one specific variant, it is mislabeled and unusable if it is really a different variant.  Many vaccines are now polyvalent, targeting multiple viruses or multiple variants within a single virus.  This adds a whole new dimension of not only have the correct set of vaccines been blended together, but is the fraction of the whole for each one within defined bounds.  As RNA products, there is also the question of whether the RNA is what was intended and no mutations have arisen during propagation of the plasmid.

Similarly, was the correct uridine analog used in production?  In vitro transcription may generate undesirable products, such as double-stranded forms of the intended product.  How much of these are present?  What fraction of the transcripts are capped? Are the RNAs full length or are there partial or degraded versions present? How much plasmid is left, and is it linear or closed-circular form?  How much E.coli genomic DNA contamination is present?

Many "old school" technologies exist for many of these questions.  A standard gel can be used to assess the length distribution.  Sanger or short read sequencing can be used for sequence verification - though Sanger will be a poor choice for multivalent designs.  HPLC may be used for a number of the questions.  But typically each assay asks a single question, and often with significant constraints. For example, if a problem is discovered in a multivalent vaccine in which there are out-of-spec shorter RNAs present, can Sanger or short reads tell which component is degraded?  

Pfizer has published an approach using specific RNA cleavage (harking back to how Woese sequenced RNA to create the Archea hypothesis - and much before) feeding into mass spectrometry.  In some ways it looks like really short short read sequencing - some fragments are indistinguishable.  The perceived advantages are that this method can distinguish fragments with the correct uridine analog vs. those with just uridine and it can distinguish capped 5' end fragments from uncapped ones.  I've meant to do a deep dive on this for over a year after Kevin McKernan had pointed me to it; time to re-prioritize that!

ONT is proposing that Direct RNA sequencing (plus DNA sequencing of plasmid batches) can be used to build a single assay to test nearly all - if not all - of the final drug product and standard DNA sequencing for assessing batches of circular or linearized plasmids.  As noted in my piece on ElysION and TraxION, this sort of "applied market" would be very appealing to ONT in terms of providing a steady source of revenue.  Direct RNA is the only currently marketed sequencing approach that can look at the modified bases, potentially giving ONT a large edge.  Many of the questions of interest are better answered with long reads - the distribution of RNA species lengths, which RNA species are which length - giving any long read platform an edge.  Should there be a problem, long read sequencing can quickly identify correlations between different anomalies.  

Of course, this does require levels of precision and accuracy.  Data was presented suggesting that minor variants can be detected at around 1% frequency.  Improved algorithms for poly-A length determination appear to enable very precise determination.

ONT dreams of covering more angles.  For example, nanopore sequencing on its own probably can't determine whether the 5' cap structure is present. But, with some sort of pre-processing - perhaps resembling Cappable-Seq/Recappable-Seq, it may be possible to tag either correctly capped or non-capped messages.  Similarly, it may be possible to differentially tag single stranded and double-stranded RNA

In terms of scale, Direct RNA sequencing in the current ONT protocol cannot be barcoded.  For huge infectious disease batches that may not be an issue; for small personalized cancer vaccine batches cost may be more of an issue.  Flowcell washing may be one solution, or ONT may be driven to enable barcoding (there are apparently external protocols for this).

How big a market will RNA vaccines be for ONT?  That is of course the big question.  mRNA vaccines seem to be here to stay, but how many more vaccines will be launched?  Delivering other therapeutics by mRNA is still an unproven market.  If mRNA delivery turns out to be a growth market, ONT can ride that wave.  If it remains a niche market, there's still gain for ONT but not what will drive them to profitability.  Lacking a reliable crystal ball, everyone must simply wait to see how this unfolds.

Conflict of Interest Disclosure / humble brag / me pretending to do Business Development.  I am (still!) employed by and hold stock in Ginkgo Bioworks. During the pandemic Ginkgo Bioworks developed a new fermentation process for producing Vaccinia Capping Enzyme (VCE).  This process is ten-fold more productive than the baseline process.  Ginkgo licensed this process to Aldevron, which is now owned by Danaher.  So production of mRNA therapeutics with capping using VCE may, through an opaque process, benefit me financially.  Little to no evidence of that so far, but it could happen!  And if you have a fermentation process that could be tuned up, feel free to reach out to me!

ONT T2T Genome Bundle: Hot New Thing or Flash in Pan?

Last month at London Calling, Oxford Nanopore announced a consumables and reagent bundle which enables generating six telomere-to-telomere (T2T) human genomes for $4K each.  Even in the very friendly audience at London Calling, there was some skepticism over the market viability of this offer - how much would it really drive sales?  T2T human genomes really only became possible in this decade.  The first examples of T2T chromosomes generally used a mix of different technologies, often including PacBio HiFi, ONT Ultralong and BioNano Genomics mapping information.  What ONT is proposing is the ability to routinely generate T2T genomes using only ONT data.  

ScalePlex: Easing High Sample Count 3’ scRNA Sequencing

Scale Bioscience officially rolled out today - their rep was already talking about it at the Boston Single Cell Symposium I attended yesterday - a new cell indexing reagent called ScalePlex to streamline single cell 3' RNA sequencing of multiple samples.  

Aftermath

I have multiple drafts of posts trying to finish up my London Calling items and then a long list of ideas in various stages of gestation - and been dangled a new tech update under embargo.  But today, I'm on a mission - to help my now former colleagues.  My employer, Ginkgo Bioworks, has executed an approximately 25% layoff.  I survived the cut, but the list of talented, wonderful people who have been cast away is long and covers a wide range of talents.  You really could start multiple quality small biotechs with these new unemployed people.

I've been laid off twice before and it's miserable.  I was lucky each time and had only a short period of unemployment - but biotech was doing well each of those times.  The industry is in a serious slump right now, with many companies cutting back and some closing altogether.  Even large companies are slashing away - Takeda is setting free over 600 employees here in Boston - perhaps some are remnants of the Millennium acquisition.  Far too few companies are being created.  

So if you have leads on open positions, I am listening.  You can leave comments, email me (keith.e.robison on Gmail), connect on LinkedIn, DM on Twitter, etc.  I've never received a message by carrier pigeon, but if that's you're style I won't object.  All will be passed on to a Ginkgo alumni community.

As a meme I saw put it "this too shall pass - perhaps pass like a kidney stone, but it shall pass".  The long-term societal upside from biotechnology is too great for this to be anything but a temporary dip - but temporary can be a very long time.

CariGenetics: Breakthrough Breast Cancer Genetics in the Caribbean - but Also a Template for ONT Clinical Push?

London Calling isn't nearly as exhausting as AGBT, but the first day of talks is packed and then follows with the social event that goes late - this year with CEO Gordon Sanghera living out his dream of being the frontman for a band.  Then if you'd like you can follow the crowd to a pub to drink on ONT's tab (that and crashing the ONT wrap-up dinner is the extent of my drawing personal benefit from ONT, contrary to a commenter on the prior piece who wrongfully believes they fund my LC expenses), and when that pub closes to another one (I peeled off after the first pub).  So one can be a bit draggy heading into the second morning, but that was solved quickly by CariGenetics CEO Dr. Carika Weldon, who wowed with an exuberant strut down the central runway to a lively calypso beat - and then wowed everyone further with a stellar presentation.  She also gave the lunchtime Product Demo talk (alas, I can't find that talk either on YouTube or in Nanopore Community) in the central product area, filling in some colorful details on her young company's early travails - all resolutely conquered.

ElysION vs. TraxION: Divergent Shots at Applied Market End-To-End Automation

London Calling was a particularly good opportunity to take stock of Oxford Nanopore's progress to a "fire-and-forget" sample-to-answer solution for "applied markets" such as food safety, public health and biotherapeutics quality control.   ElysION (formerly Project TurBOT) and TraxION represent very different approaches targeting different subsets of this broad market opportunity - and I heard from some interested parties that neither is quite what they want.  That doesn't mean they aren't right, but it does mean ONT may need to think of more approaches.

The broad concept is to have a a device that takes some sort of biological input, with minimal to no upstream processing, and performs all necessary steps so that nanopore sequencing data emerges from the instrument, with no human intervention after the run is set up.  ONT is envisioning these being placed in clinical labs, public health labs, biotherapeutic quality control labs, etc.