Archive for the ‘Uncategorized’ Category.

Base4 Innovation Ltd

Following on from my list of sequencing companies I wanted to delve into Base4 next. Below are my brief notes on their business and technology.

Business

If you’re not really interested in the business stuff, skip this and head to the tech section below. Suffice it to say they’re a series A/B stage DNA sequencing startup and have received about 21M GBP in total investment.

The company was incorporated in October 2007. Being a UK company there’s quite a bit of information at companies house [3].

Investors appear to include: Longwall Ventures, Meridian Corporate Finance, Oxford Technology Enterprise Capital, Royal Society Enterprise Fund, Torteval Investments Ltd, and Amadeus RSEF. Amadeus RSEF I think stands for Royal Society Enterprise Fund, and possibly indicate that this investment is associated with that fund, rather than the main Amadeus fund.

Companies house accounts list 36 staff in 2016 [4] , with a burn rate of about 3M GBP a year. They had around 1.6M GBP in cash. The accounts state “to meet the company’s ongoing cash needs…additional funding will be required…within the next 12 months”.  And their site states they received 5M GBP in November 2017 [1].

Glassdoor reviews [2] for the company contain some entertaining gossip. It looks like there has been some internal restructuring at the company, but with the raise last year hopefully things have settled down.

Technology

The image below shows the basic mechanism they present on their website. The essential idea is pretty straightforward. Take single molecules of DNA, use pyrophosphorolysis to pull off individual nucleotides. The individual nucleotides then get encapsulated in microdroplet reaction vessels where they undergo a reaction which allows then to be detected by a fluorescent signal. My guess would be that the idea is that this might result in longer reads as opposed to Illumina-SBS sequencing. It seems doubtful to me that this process might result in higher accuracy reads.In addition to this method they appear to have patents covering plasmon nanopores, which I’ll cover below. First I’ll cover each part of the process above in a little more detail.

First they need to remove individual nucleotides. The normal way of doing this would be to use an exonuclease. Which is an enzyme that cleaves off bases. Others have proposed using exonucleases for single molecule sequencing (including Oxford Nanopore’s original approach). The patents don’t very clearly state what they’re doing (when are patents ever clear?) but they appear to be using an enzymatic approach, I guess either an exonuclease or a polymerase with some exo activity. The sequencing template is attached to a surface and the nucleotides get chewed off.

They then get stocastically captured in droplets (so there will we a lot of empty droplets, and quite possibly some droplets containing multiple bases). These then get transported through a fluidics system (though a recent patent suggests they might also just be arrayed on a surface [5]).

At this point they have single nucleotides in little reaction vessels, presumably with some reagents. The next stage is therefore to determine which nucleotide is in each droplet. The website currently refers to a cascade reaction and fluorescence, but patents also refer to Plasmon resonance [6].

The cascade reaction appears to work by having the single nucleotide bind to a capture site on an oligo [7]. A substitution-dependent restriction endonuclease comes in and cuts the oligo depending on whether the nucleotide is present or not. The now cut oligo can now be processed by a double-stranded exonuclease. This releases more single bases, hence producing a cascade reaction.

In the process of the above cascade reaction, fluorescent labels are activated. The exact process isn’t described (that I can see) but it could be that a quencher is removed or something similar. Like most patent portfolios, this is just one suggested mechanism… but I’d guess this is the cascade reaction mentioned on the website. That about wraps it up, by detecting the fluorescence (I assume they need multiple labels/lasers) they can call the DNA sequence. There are also patents referring to methylation detection but I’ve not taken the time to read these yet.

As a final note, a few of their patents refer to plasmon nanopores. This appears to be unrelated to their microdroplet sequencing efforts, and for an early stage company it seemed odd to be experimenting with such a radically different approach. The patents contain SEM images, suggesting that some real work has been done on this system. The approach is obviously also somewhat related to the Armonica approach previously discussed. SEM images below.

That about wraps it up for Base4. There’s some interesting tech, and a pile of patents which may no doubt yield further insights. If you’d like to discuss Base4 further (or sequencing in general) leave a comment below or email me (new at sgenomics dot org).

Notes

[1] http://www.base4.co.uk/news/base4-nov17-funding/

[2] Glassdoor reviews (selected mostly negative review containing interesting gossip):

Review 1

Pros

Good people from all disciplines, possible to learn a lot by mixing with people from different backgrounds.

Cons

Micromanagement from senior staff, some of whom have no formal qualifications in science. The CSO left and has not been replaced, leaving scientific oversight in the hands of those less qualified. Very high staff turnover.

Advice to Management

Trust those who you’ve hired, don’t micromanage. There’s no point bringing in postdoctoral level scientists then treating them like technicians.

Review 2

Pros

You can enjoy personal trainer sessions, playing anytime table soccer, free food and drinks.

Cons

The biggest problem is that you need to bare random moody, bully, disrespectful and unprofessional behavior of the CEO who, in addition, has no scientific qualifications/competences.
You might end up to go, everyday, at work with a feeling of terror because there is great probability that the CEO can be mad at you for any reason.
They promised to work in a transparent environment.. but the reality was different.. I discovered bunch of exaggerations and sometimes lies.
The management hires nice and qualified people .. but a great percentage leaves the job after 6-12 months and this slows down the project you work on.

Review 3

Pros

Shiny on the outside. That’s about it. I would not read too much into the veneer of things working smoothly.

Cons

If you like working for a schoolyard bully that has no regard for his employees then go for it.
If you want to do something complete irrelevant to what you were promised before getting the job and if you like to have an adjustment period of about 5 seconds, then again, go for it.
If you like being proud that your are simply stumbling along with no clear vision from the management, then again, fell free to join.
In your shoes I would probably try to work in a different building.

Review 4

Pros

It is a fairly small company with approx. 15 people. It has no strict regulations and you have relatively flexible working time.

Cons

If you are not a DNA specialist or a chemist, do prepare that you need to explain everything from fragment to your manager as they know nothing but they oddly have the confidence that they can recruit people.

Interview Question

After applying online via cv, covering letter and a questionnaire, was asked for Skype interview. One of the most bizarre interviews I’ve ever had. Was asked a single question – ‘why I don’t you have a Ph.D. then?’ to which I replied I’d wanted to get into industry at which the CEO rather abruptly ended the interview.

Was left with the impression that Base4 is very much led by the CEO’s gut instinct and this could be a difficult place to work unless you are a good personality match with him.

[3] https://beta.companieshouse.gov.uk/company/06389614

[4] Interestingly in the funding news in 2017 they state 32 employees. By guess would be that prior to the round there was a hiring freeze, and a few people left.

[5] “Whilst the method described above can be carried out by creating and manipulating a stream of the droplets dispersed for example in an immiscible carrier medium such as silicone oil, we have recently found that the method can advantageously and more effectively performed by printing the droplets directly onto the surface of a substrate as they are formed.” https://patentimages.storage.googleapis.com/15/95/c0/60260db5a928fa/EP3115109A1.pdf

[6] “1. A method for determining the sequence of nucleotide bases in a polynucleotide analyte, the method comprising steps of: (a) generating a stream of droplets at least some of which comprise both (1) a single nucleotide base and (2) colloidal metal particles capable of undergoing plasmon resonance, and (b) irradiating each droplet with electromagnetic radiation to (1) cause the metal particles contained therein to undergo plasmon resonance and (2) the nucleotide base also contained therein to Raman scatter light at one or more wavelengths characteristic of its type. ”

[7] “(a) a first single-stranded oligonucleotide labelled with first and second regions of characteristic detectable element types in an undetectable state located respectively on the X’ and Y’ end sides of a third region comprising a restriction enzyme recognition site element including the capture site and an exonuclease-blocking site on the X’ side thereof (wherein either X’ is 3′ and Y’ is 5′ or X’ is 5′ and Y’ is 3′) and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide flanking the capture site; (2a) either (i) treating the used probe with a conventional or nicking substitution-dependent restriction endonuclease to cut the first oligonucleotide strand at the recognition site if and only if the single nucleotide captured comprises a nucleobase which is substituted or (ii) treating the used probe with a conventional or nicking substitution-sensitive restriction endonuclease to cut the first oligonucleotide strand at the recognition site if and only if the single nucleotide captured comprises a nucleobase which is unsubstituted; (3) digesting the first oligonucleotide strand of the used probe with an enzyme having double-stranded exonucleolytic activity in the X’-Y’ direction corresponding to the first oligonucleotide to yield detectable elements derived from either the first region, the second region, or the first and second regions in a detectable state and a single-stranded fourth oligonucleotide which is at least in part the sequence complement of the first oligonucleotide; (4) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe; (5) repeating steps (2a), (3) and (4) in a cycle and (6) detecting the detectable elements released in each iteration of step (3) wherein if the endonuclease employed is of the conventional type the second or third oligonucleotide includes an endonucleolysis-directing linkage at or close to its X’ or Y’ end respectively.”

[8] “Further information about the pyrophosphorolysis reaction as applied to the degradation of polynucleotides can be found for example in J. Biol. Chem. 244 (1969) pp. 3019-3028. The enzyme which is preferably employed in this pyrophosphorolysis reaction is suitably selected from the group consisting of those polymerases which show essentially neither exo- nor endonuclease activity under the reaction conditions. Examples of polymerases which can be advantageously used include, but are not limited to, the prokaryotic pol 1 enzymes or enzyme derivatives obtained from bacteria such as Escherichia coli (e.g. Klenow fragment polymerase), Thermus aquaticus (e.g. Taq Pol) and Bacillus stearothermophilus, Bacillus caldovelox and Bacillus caldotenax. Suitably, the pyrophosphorolytic degradation is carried out in the presence of a medium which further comprises pyrophosphate anion and magnesium cations; preferably in millimolar concentrations. ” https://patents.google.com/patent/WO2014167323A1/en?oq=WO2014167323+

Armonica Technologies LLC (now Inc)

In my last post I put together a list of 41 companies which either have a DNA sequencing platform on the market, or in development. There is really only one company that dominates this market (Illumina) so most of the players are either niche plays or startups. Armonica is one such early stage startup.

Business

They appear to have raised 1.5MUSD [1]. Before looking into the technology, there are a number of things that stand out about Armonica. Firstly it’s an LLC, typically the received wisdom for creating a venture funded tech startup is to create a Delaware C-Corp. From what I can tell, creating an LLC could be problematic for traditional investors (who might want preferred shares for example) [2] (appears to now be listed as Armonica Technologies Inc. [13]).

The next thing that stands out is the list of “Board Of Directors/Executive Leadership”.

There are 5 people on this list [3], but of them only Steven Brueck (CSO) and Scott Goldman (CEO) appear to be part of the company (the rest are investors). Sally Corning is listed as co-founder, and a partner at a fund, which seemed odd to me.

A LinkedIn search shows Scott and one other senior consultant. The CSO is a university professor so most likely doesn’t do much at the company day-to-day. An SBIR entry suggests they have 23 employees which seems surprisingly high [12].

Overall, it seems to be seed stage, the company was founded in 2015 [4].

Technology

Armonica is an optical nanopore DNA sequencing company. Optical nanopore approaches use a nanopore to control the motion of a strand of DNA but an optical readout (as opposed to direct electrical detection [5]). Armonica are not the only company to propose the use of an optical nanopore, Quantapore have presented a protein nanopore, FRET based concept too. But the method suggested in Dr Brueck’s patents [6] and paper [7] differs significantly. Firstly, the fabrication methodology they’re suggesting is quite novel, and secondly the optical readout is label-free (using surface plasmonic resonance).

First up, fabrication. There are a number of novel features here firstly they’re using interference lithography to define their features. Interference lithography is one technique that lets you beat the diffraction limit and create features smaller than the wavelength of light. It does this by using the interference between two coherent light sources to define the pattern. Pretty neat! The downside is of course that you are limited to periodic structures, but that seems fine for nanochannels and they present SEM images with channels as small as 100nm.

After using interference lithography to lay down photoresist they then lay down Silica nanobeads, after which they heat the whole thing, baking out the photoresist and sintering the silica to create the final structure. Neat! The exact advantage of using nanobeads over other deposition methods is less clear to me (I’m no expert) and some quick googling brings up some alternative methods for creating enclosed nanochannels [8]. It would be interesting to dig further into this aspect of the technology, and contrast the various approaches here.

With nanochannels in place, they can DNA through them (see below) . This is where their paper finishes up. The Silica is transparent, so they can image YOYO stained double stranded DNA right through it. They note that in the small (100nm) channels, it’s harder to get the DNA moving through, and that it moves more slowly (driven only by capillary action here).

DNA imaged in nanochannels from [7].

The above is based on published work out of Brueck’s lab. The patents (and brief description on the website) mix things up a bit, and describe a tortuous nanopore. In the patent tortuous nanopores are rather aptly described as “complex, 3D paths, similar to the spacings and open paths created when oranges are piled up in the local supermarket”. So, the idea is rather than precisely defining a nanopore, you can stochastically define them with these piles of silica nanobeads. You can also use a more traditional deposition method to (somewhat selectively) close apertures in this “tortuous nanopore”. This also seems like a rather neat idea. The tortuous nanopore concept potentially provides a method for slowing translocation (generally an issue with solid state nanopore systems) and for generating small apertures than available with traditional fabrication processes. It makes me wonder if the system couldn’t be used for ionic current detection (or perhaps there is too much stray ionic current, or other issues).  From what I can tell the system uses a bias voltage to drive the strand through the pore.

The base detection process is probably the least detailed part of the patent. My understanding is it essentially states that they’ll use surface plasmon resonance, and that there are a bunch of ways of doing that. Using SPR for DNA sequencing isn’t a new idea, and a quick google brings up grants for as early as 1999 [9]. But I would have to do more reading to know how practical it is here. Interestingly another company (Redondo Optics [10]) has an SBIR grant [11] for using SECAR SPR for DNA sequencing. The CEO of Redondo optics is also on the Armonica SAB. With more time this would be an interesting avenue to follow up.

For the moment however, I leave this brief review here. Overall the Armonica technology seems to have a number of interesting novel aspects. If you have any further insights into the technology, or would help to chat about DNA sequencing drop me a line (new at sgenomics dot org), or leave a comment!

 

[1] https://www.abqjournal.com/1050880/unm-technology-accelerates-genomic-sequencing.html

[2] https://mashable.com/2016/01/27/startup-business-structure/#eSxUGPb_qGqX

[3] Currently listed on the website are:

David Blivin, MBA Managing Partner, Cottonwood Technology Funds

Steven R. J. Brueck, Ph.D. Chief Scientific Officer, Armonica Technologies

Sally Corning, MBA Co-Founder & Partner, Sun Mountain Capital

Scott Goldman, MBA President & Chief Executive Officer, Armonica Technologies

Waneta Tuttle, MBA, Ph.D. Fund Manager, Tramway Venture Partners

[4] SEC Filing https://www.sec.gov/Archives/edgar/data/1714729/000171472917000001/xslFormDX01/primary_doc.xml

[5] Most other nanopore companies propose detection through the blockage of ionic currents, or tunneling currents.

[6] US9927397: https://patentimages.storage.googleapis.com/56/92/ad/2b5ef43d1f4c47/US9927397.pdf

US20160377590A1: https://patentimages.storage.googleapis.com/d1/8a/16/0bc8e6aafcf270/US20160377590A1.pdf

[7] http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.172.7987&rep=rep1&type=pdf http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.172.7974&rep=rep1&type=pdf

[8] https://www.princeton.edu/~chouweb/publications/148%20Austin_Scanning%20the%20Controls_IEEE%20Nanotech_2002.pdf

[9] http://grantome.com/grant/NIH/R21-HG002102-01

[10] http://www.redondooptics.com/

[11] https://www.sbir.gov/sbirsearch/detail/1031029

[12] https://www.sbir.gov/sbirsearch/detail/1490917

[13] https://www.sec.gov/Archives/edgar/data/1714729/000171472917000001/xslFormDX01/primary_doc.xml

DNA Sequencing Companies (July 2018)

Below is a list of 40 companies who either have a DNA sequencing platform on the market, or where there’s public information that they are developing a DNA sequencing/mapping platform. There are a few cases where it’s not clear if the company is still active, I’ve tried to note this. If have any information on these companies, know of any others, or would just like to chat about sequencing companies/the sequencing market in general, feel free to email me (new at sgenomics dot org).

Disclosure: I’ve worked at a few of these companies, all the information presented here is based on public information.

Due to the width of the table, it’s best viewed on desktop.

Name Further Info Status Detection method Single mol? Chemistry Location
Armonica Technologies LLC Company Website Seed? Optical-nanopore Yes Direct (label free) New Mexico – USA
Base4 Company Website Series A+? Microdroplet/optical (+nanopore) No Pyrophosphorolysis Cambridge – UK
BGI (Complete Genomics) Company website Acquired Optical No SBL/SBH Mountain View – USA
BioNanomatrix Company Website Series D Optical Yes hybridized? tags San Diego – USA
Caerus Molecular Diagnostics Company Website Seed – 2014 Optical Yes SBS? Mountain View – USA
Centrillion Company Website Series B Optical No SBS on Array Palo Alto – USA
Cygnus Biosciences News Article Series B Optical No SBS Beijing – China
Depixus (was Picoseq) Company Website Series A Unzipping force Yes SBH/Interference Paris
Direct Genomics Company Website Unknown Optical Yes SBS Shenzhen – China
DNAe (DNA Electronics) Company Website Series A + X? ISFET No SBS (unlabelled) London – UK
Electronic Biosciences Company Website Seed+? Protein-nanopore Yes Direct (label free)? San Diego – USA
ElectroSeq Genomeweb Article Pre-seed? ISFET No SBS? New Mexico – USA
Eve Biomedical Funding note Seed, Dead? Carbon Nanotube – FET Yes SBS? Mountain View – USA
Genapsys Company Website Series B Undisclosed No SBS? Redwood City – USA
Genedra Biotech Ltd Company Website (dead) Dead? Optical Unknown SBS Beijing – China
Genome Surveillance, Inc. Online note Pre-seed Unknown Yes? Restriction map? Wisconsin – USA
Illumina Company Website IPO Optical No SBS San Diego – USA
iNanoBio Company Website Seed? Nanowire – FET Unknown Unknown Arizona – USA
inSilixa Company Website Series A ISFET + other No SBS (unlabelled) Sunnyvale, CA – USA
Intelligent biosystems (Qiagen) Company Website Acquired Optical No SBS? Netherlands
Ion Torrent (ThermoFisher) Company Website Acquired ISFET No SBS (unlabelled) Massachusetts – USA?
Lasergen (Agilent) Company Website Acquired Optical No SBS Texas – USA
Lightspeed Genomics Company Website Acquired? Optical Unknown Unknown Santa Clara – USA
Molecular Research Limited (Mobious) Company Website Seed? Alive? Molecular Resonance Sequencing Unknown Unknown Exeter – UK
Nabsys 2.0 Company Website Series E+ Solid-state nanopore Yes hybridized tags Rhode Island – USA
NanoString Technologies Bio IT World Article Series E+ Optical No hybridized tags Seattle – USA
Northshore Bio Company Website Series A Solid-state nanopore Yes Direct (label free) Camas, WA
Omniome Company Website Series A+ Undisclosed Undisclosed SBB San Diego – USA
OpGen Company Website IPO Optical Yes Restriction mapping Maryland – USA
Oxford Nanopore Technologies Company Website Series I+ Protein-nanopore Yes Direct (label free) Oxford – UK
PacBio Company Website IPO Optical – ZMW Yes SBS Menlo Park – USA
Personal Genomics/CrackerBio Video Unknown Optical Semiconductor No SBS? Taiwan
Quantapore Company Website Series C Optical-Nanopore Yes All bases labelled Menlo Park – USA
Quantum Biosystems Company Website Series B Nanopore/gap Yes Direct (label free) Osaka – Japan
QuantumDX Company Website Series A Nanowire – FET Yes/No Hybridized tags Newcastle – UK
Roche (Genia Technologies) Company Website Acquired Protein-nanopore Yes SBS Santa Clara – USA
Roswell Biotechnologies Company Website Seed/Series A? Nanogap? Yes Direct? San Diego – USA
SeqLL Company Website Series A Optical Yes SBS Massachusetts – USA
SingularBIO Company Website Series A? Unknown Unknown Unknown San Francisco – USA
Singular Genomics Note Unknown Unknown Unknown Unknown San Diego – USA
Two Pore Guys Company Website Series B? Solid-state nanopore Yes Unknown/Various Santa Cruz – USA
XGenomes Company Website Seed? Optical Unknown Unknown Boston – USA

SBS: Sequencing-by-Synthesis

SBH: Sequencing-by-Hybridization

SBB: Sequencing-by-Binding

Inheco Control 96 + CPAC Ultraflat Notes (TEC/Peltier)

I picked up this Peltier system on eBay a while back. I guess it’s for heating eppendorfs as part of some larger system.  The device is an example of the sub-contractors/assemblies all the way down mentality  you see in so much industrial equipment. The control unit uses a Watlow 96 PID controller, and a Watlow LSTW driver, which they’ve put in an INHECO branded chassis.

When I received it the controller seemed to be misconfigured. The TEC wouldn’t heat, and could not be set beyond 50C. Datasheets for the CPAC Ultraflat state that it should be able to go to 70C. So I needed to do some fiddling to figure out what exactly was going on.

The LSTW driver doesn’t seem to be documented anywhere on the web, I couldn’t find anything similar on the Watlow website either, so it’s possibly some kind of custom OEM module they have… However, as far as I can tell it’s just an H-bridge taking a 12v input.

It’s controlled by two pairs of inputs from the PID controller, one sets the output positive, the other negative. So from what I can tell it’s using purely digital control.

Looking at the PID controller itself, output 1 and 2 are wired up. As is the RTD temperature sensor:

The Watlow control interface is a bit of a pain, but it’s functional enough once you know what’s going on, and I configured inputs/outputs as follow:

Sensor: Wired for 2 wire RTD 100Ohm Platinium.

Outputs 1 and 2 on, switched DC open collector output.

Output 1 set to control heating.

Output 2 set to control cooling.

I also set the control limits to +/- 100C. I’ve pushed it to 90C and it seems to work, though it takes a while to get up to temperature. Not sure what effect running it out of spec will have. Quick thermal image with a FLIR one below. When I can find my temperature probes I’ll probably try and do some more tests… That’s it for the moment, more pics below.