My contribution to the increasing entropy, and eventual heat death of the universe
Archive for July 2018
I picked up a couple of minispin lab centrifuges on eBay. One was working, one faulty. I pulled apart and took some pictures for reference (below). Unfortunately it looks pretty dead. In particular IR2136J 3-phase driver is completely blow. I’ve ordered a replacement and we’ll see how that goes.
However in case they’re of use to anyone else, reference pics below:
I picked up a Corning 420D on eBay, the device was listed as not powering up and was relatively cheap so I figured worth a risk. It turned out to be a very simple repair, but I figured I’d write it up anyway… perhaps the reference images will be of use to someone.
The device indeed showed no power LEDs, but the hotplate was heating up… Taking a look inside you can see that the design is pretty simple. All through-hole, there’s what looks like an 8bit MCU of some description. The stirrer has a optical interrupter sensor on it, which is attached directly to the PCB.
There were no obvious issues on the top side of the PCBs so I removed the main PCB and discovered the ugly mess below… I guess there had been some lab spills…
Cleaned things up with some IPA and touched up some of the joints, but the device still didn’t work (unsurprising). So I started probing around. There’s a 74HCT7541 on the front which was a convenient location to measure digital supply voltage levels… VCC was less than 2V and fluctuating…
I probed the transformer this was putting out ~10v AC and was being rectified… But there was nothing coming out of the regulator (probably a 7805 but I didn’t even get round to checking). Turns out the trace was broken just before the regulator, when I bridged this everything started working… Sorry not a very exciting repair!
You can see the bodge in the picture below (I used a small piece of wirewrap wire). I could do with cleaning the PCB up a bit more, but I’ll leave things as they are for the moment.
I never quite know where to place the BGI (originally Beijing Genomics Institute). Are they a sequencing service? Are the a instrument vendor? Are they a business at all? Or are they a research institute? At times they seem like all these things, and none of them. My clearest personal memory is from one of the Cold Spring Harbor conferences. The moderator announced that before our next talk a representative from the BGI would like to make a brief statement. The BGI representative stood up and read a pre-prepared statement announcing that the BGI would sequence 1000 plant an animal genomes… it was an ambitious project but clearly one that the BGI were capable of accomplishing (using Illumina machines at the time). But the delivery seemed weird. Usually these large projects are coordinated through an international consortium of researchers. The BGI just stood up and stated that they were going to do all this themselves… it kind of sounded like a declaration of war, and indicated to me that the BGI is something other than a traditional research institution.
At present they a massive fleet of Illumina machines, their own commercial instruments employing two different sequencing technologies, a sequencing as a service business, a clinical NIPT test, 1000s of researchers, and offices in 4 countries. So what exactly is the BGI anyway?
bgi.com in 1998
I figured I’d start by trying to go back to the beginning. The BGI was founded in 1997, and appears to have been developed out of China’s desire to take part in the human genome project. bgi.com wasn’t owned by the BGI in 1998. genomics.cn was most likely, but the earliest capture in the waybackmachine is from 2008, it’s quite sparse and talks of them having Illumina Genome Analyzers, ABI Solids and 454FLXs (it’s notable that only the Illumina range of instruments still exists). But it’s otherwise not very instructive.
The BGI history page, tells us a little more stating that “On July 14, 1999, BGI was founded with the mission of 1% of the human genome for the International Human Genome Project.”. In China research funding comes from the Ministry of Science and Technology . I would imagine funding came from the “973” program. Which was China’s basic research program until 2017, when it was supposed to be replaced by something else.
Early versions of the Chinese wikipedia page on the BGI state that after the completion of the human genome project the BGI relocated to Hangzhou in exchange for local government funding, and then in 2007 they announced that they were to relocate to Shenzhen to establish China’s first private non-profit research institution, in 2008 BGI Shenzhen was approved by the Shenzhen Municipal Government to become a public institution. So from what I can tell they started off firmly as a non-profit research institution and remained so until at least 2008. What’s less clear to me is what kind of entity this was (non-profit? state owned? private company?).
2010 seems to have been the turning point for the BGI. They received a 1.58B USD line of credit from the China development bank [1], and funds from Shenzhen Capital Group [2]. It also looks like Taikang Life Insurance [2] and Sequoia may be an investors [4]. Then in 2013 after a long and seemly slightly painful process they acquired US DNA sequencing startup Complete Genomics [3]. Finally on the 14th July 2017 they were listed on the Shenzhen stock exchange (company 300676) completing their transition in to a somewhat surprisingly highly commercial research institution.
The Chinese wikipedia page now lists 6 business units: BGI Research Institute, BGI Technology Service Co., Ltd. , BGI Health, BGI Agriculture, BGI Genetics Institute and BGI Cloud Computing.
Glassdoor reviews for BGI Shenzhen seem pretty reasonable (mostly complaining about the pay). Glassdoor reviews for Complete Genomics post acquisition seem to indicate that much development work was transitioned to China, and tantalizingly that the “omega sequencer project” was killed.
That about wraps it up for the business side of things. I will continue to add information as I come across it (not sure what information Chinese companies need to make available, but if anyone knows I’d be most interested in revenue projections etc.). I’ve covered the technology they acquired from Complete Genomics and plan to cover other aspects of their business too.
[1] https://www.technologyreview.com/s/511051/inside-chinas-genome-factory/
[2] https://www.crunchbase.com/organization/bgi-2/funding_rounds/funding_rounds_list
[3] http://www.genomics.cn/en/news/show_news?nid=99460
[4] https://www.sequoiacap.com/china/en/companies/
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.
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]. This may indicate that further fundraising would need to take place in 2018.
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.
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. It seems likely that this may relate to their collaboration wit Hitachi that was reported in 2013 [9].
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).
[1] http://www.base4.co.uk/news/base4-nov17-funding/
[2] Glassdoor reviews (selected mostly negative review containing interesting gossip):
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.
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.
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.
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.
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+
[9] https://www.genomeweb.com/sequencing/base4-hitachi-developing-single-molecule-nanopore-based-sequencer
“Privately owned Base4, the partners said, has developed a method of increasing the signal from the single molecule passing through the pore by using laser light enhanced by gold structures. The technology, they added, allows the signal to be read from unlabeled DNA, minimizing sample preparation.”
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