41J Blog

Business

There’s not much business information available on Caerus, they’ve received ~1.2M USD in SBIR grants [1] most recently in 2014. I can currently only find one employee on LinkedIn (one of the founders, Javier Farinas), though I can find 3 other previous employees (including the co-founder, Andrea Chow). Caerus received their first grant in 2010, and I would guess they would likely need to raise soon to sustain R&D work on their platform.

Technology

Caerus originally proposed using a method they called Millikan sequencing. This was a method measuring the charge of a strand as nucleotides were incorporated. They published a proof on concept of this system in 2015 [2], however they also announced that they were abandoning the approach around this time [3].

The Millikan sequencing approach was pretty interesting. In the proof of concept they present a system where a bead covered with multiple DNA templates is suspended between two electrodes.

They apply an AC bias voltage and can see the bead moving under the changing electric field (the DNA being negatively charged). The proof on concept uses sequencing by synthesis. As bases are incorporated the charge on the bead changes and these incorporations can be detect by changes in the motion of the bead (they use a velocity measurement).

It’s pretty neat that they could use natural, unlabeled nucleotides (which could potentially lead to longer reads). But they have apparently abandoned the approach. Because they used amplified DNA, they would be subject to the same phasing errors that limit read length on other platform. Ion torrent also used unlabeled bases and I guess they figured the competitive advantage was too small. Having an optical sequencing technology that uses unlabeled bases is kind of neat however (but I guess also similar to 454s platform).

After abandoning the Millikan approach, they started working on something called “activator sequencing”. This method is covered in their 2014 patent [4].

The patent states “the methods use an enzyme to covert a product produced from a sequencing reaction into many copies of a readily detectable report molecule”. Essentially what they appear to be suggesting is single molecule sequencing [5] system where incorporation activates an enzyme which in turn creates many copies of a detectable (e.g. fluorescent) molecule.

It seems like an interesting idea, but at the single molecule it’s possible that engineering efficient enzyme activation might be problematic. It will be interesting to see how things progress!

Notes

[1] https://www.sbir.gov/sbirsearch/detail/12662

[2] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4560655/

[3] https://www.genomeweb.com/sequencing-technology/caerus-molecular-explores-activator-sequencing-long-single-molecule-reads-low

“For amplified DNA, they did show proof-of-principle of sequencing, which they published last month in Analytical Biochemistry. However, it did not make sense to develop that approach commercially. “It was pretty evident that we were not being able to catch up to the rest of the field using that method,” Farinas recalled, noting that Ion Torrent came out with the PGM around the same time, which offered many of the same benefits, including label-free sequencing, that Millikan sequencing promised. “Basically, they had a commercial product and we had some preliminary data,” he said. ”

[4] http://www.freepatentsonline.com/20160068902.pdf

All Patents:

Click to access 20120220486.pdf

http://www.freepatentsonline.com/y2016/0068902.html
http://www.freepatentsonline.com/y2010/0112588.html
http://www.freepatentsonline.com/y2011/0059864.html

[5] The patent also suggests they the scheme could be used with colonies (clusters).

It might seem odd for Bionano to be on a list of DNA sequencing companies given that Bionano have not publicly discussed the developing a DNA sequencing platform. Bionano are instead focusing on genome mapping. Mapping technologies give long range information on the structure of the genome (megabases, at >100bp resolution). This allows you to detect large scale structural variation in a genome (such large scale changes are associated with cancer for example). In contrast to this, most current DNA sequencing technologies focus on accurate short range information, providing single base resolution reads of about 100bp.

However mapping and sequencing exist in a continuum. As sequencing read lengths increase (into the mega base range with some approaches) they can more easily address issues of structural variation. Similarly, as the resolution of a mapping technology decreases (ultimately to single base resolution) it turns into a long read sequencing technology.

Because of this technological continuum, I’ve included mapping companies that have their own unique sensing system. And in this post I discuss Bionano Genomics.

Business

Bionano genomics (originally Bionanomatrix) was founded in 2003, to date they have raise 132.1M USD (according to crunchbase) from a number of investors [1], they also received about 3M USD in SBIR grants [3]. Their first commercial instrument launched in 2012. They now appear to be filing for an IPO seeking to raise 34.5M USD [2]. Genomeweb also reported that Bionano received 1.7MUSD in revenue Q1 2018 (net loss 3.8M). On March 31st they had 7.6MUSD in cash. Meaning without further financing they can last a couple for quarters. In 2017 total revenue was 9.5MUSD. So the 2017 average revenue per quarter was a little higher than Q1 2018s. They currently have 65 employees (28 in research).

Technology

Bionano’s devices were at least partially developed from work undertaken at Lund University and Princeton. Patents refer to a 2004 paper co-authored by Bionano Genomics founder Han Cao [5]. The paper shows 100nm nanochannels:

In this paper double stranded DNA is driven through the nanochannel under a bias voltage. There’s a nice image of some stretched DNA:

Here is DNA is labelled with TOTO. This is an intercalating dye which labels the entire strand. The Bionano chip seems to be a development of this, and there’s a nice schematic on the Bionano website:

As you can see the chip is somewhat more advanced than that presented in the 2004 paper. In particular pillars have been added to untangle DNA. Once the double stranded DNA is linearized in the channels it can be imaged.

But just imaging stained DNA isn’t very useful. In order to create a structural map of the fragments, we need some kind of specific labelled to create a “map” of the fragment. Most typically this is performed using a nicking endonuclease. These enzymes recognize particular target DNA sequences and create small single stranded “nicks” in the DNA:

Nicking process from Bionano patent.

Fluorescent probes can then be attached to the nicked locations to allow their locations to be imaged. Using this information you can obtain a map of the DNA strand, telling you the relative locations of the nicking enzyme recognition sites. You can then compare these molecule maps against known references. By looking for large scale rearrangements, it’s possible to determine where structural variation occurs.

The system resolution is likely limited by several factors, and their site states that the Irys system resolution is 1.5 kbp or ~500nm, this would make sense as the optical resolution of a diffraction limited system.

In addition to there currently released platform Bionano do have one patent where they incorporate sequencing into their platform [4], but given this is from 2009 it seems likely that they have not pursued this approach currently. Some more digging might reveal other interesting approaches in their patents.

Notes

[1] According to Crunchbase, investors include: Domain Associates , Novartis Venture Fund , Legend Capital , Battelle Ventures , Gund Investment, LLC , KT Venture Group , Innovation Valley Partners , 21Ventures and Ben Franklin Technology Partners of Southeastern Pennsylvania.

[2] https://www.genomeweb.com/business-news/bionano-genomics-seeks-raise-345m-ipo

“Bionano also revealed that it had $1.7 million in revenue in the first quarter of 2018 and a net loss of $3.8 million, or $1.16 per share. Its R&D expenses for the quarter were $2.4 million and its SG&A expenses were $2.9 million. As of March 31, the company had $7.6 million in cash and cash equivalents.

In 2017, the company had $9.5 million revenues and a net loss of 23.4 million, or $7.66 per share. Its R&D expenses were $12 million in 2017, and its SG&A expenses totaled $14.1 million.

As of December 2017, Bionano had raised approximately $129.3 million through sales of its preferred stock. In addition, in 2016, it entered into a secured term loan facility with Western Alliance Bank, under which it has borrowed $7 million. The loan facility requires it to raise $5 million from the sale of equity securities by Aug. 3 of this year.

Bionano said that as of March 31, it had 65 employees, including 28 in sales, sales support, and marketing, 28 in research and development, four in manufacturing and operations, and five in general and administrative positions. Of its employees, 57 are located in the US and eight elsewhere.”

[3] https://www.sbir.gov/sbc/bionanomatrix-inc

[4] https://patents.google.com/patent/CA2744064A1

[5] http://www.pnas.org/content/pnas/101/30/10979.full.pdf

I’ve previous discussed the BGI Business, and the DNA sequencing technology they acquired from Complete Genomics. In this post I want to tie up a few loose ends. In particular, I was curious to see if the BGIs patents covered any other approaches. From what I can tell, their patents don’t cover other approaches in much depth, but there are a few interesting bits and bobs!

MGI Tech

MGI Technologies is a wholly owned subsidiary of the BGI Group. They’re currently looking to IPO on the Hong Kong stock exchange, and are attempting to raise 1B USD [2]. They have a number of patents assigned to them, these mostly concern themselves with sample prep methods. However there is one patent on DNA sequencer chip design [3] it looks very much like the BGISEQ50 chip shown on one of their sites:

SBS-patent?

There’s a patent from the BGI which seems a bit odd to me [5]. It essentially seems to cover a flowcell and SBS sequencing methodology that looks very much like the Illumina process. In fact, the flowcells look almost identical:

Chip from BGI patent…

Example Hiseq flowcell from [4].

It’s possible that there’s something I’m missing here, because the patent is in Chinese, and the translation isn’t great. But it’s interesting none the less. The patent doesn’t seem to cover specifics of the chemistry or imaging system, but seems to talk about the overall approach. MGI Tech also seem to have a patent covering a 2 color optical system [7], not dissimilar to the Illumina 2 color sequencing approach.

A number of other patents cover optical systems for DNA sequencers, but don’t appear to discuss a particular sequencing approach [6].

Nanopore Patent

There’s one nanopore patent [1] assigned to the BGI Shenzhen. It mostly concerns itself with a “multi-pass sequencing” approach, to reduce error rates. In this approach a “concatemer nucleic acid molecule comprising a plurality of copies of the target sequence” is used to reduce the overall error rate.

The patent also discusses “membranes of randomly distributed nanopores” as opposed to ordered arrays used in most systems. They suggest that nanopores are instead randomly distributed on a single membrane. Optionally they can be cross-linked to fix them in place. Micro-pipettes are then are then used to create a seal around one side of the nanopore. This is similar to a traditional patch-clamp approach, and I don’t quite understand what they’re getting at here. It seems like they suggesting billions of pipettes are used. This seems like a problematic system to build.

Notes

[1] Nanopore methods patent http://www.freepatentsonline.com/WO2016077313A1.pdf

[2] https://www.bloomberg.com/news/articles/2018-04-25/china-dna-giant-s-equipment-arm-said-to-seek-1-billion-funding

[3] Chip design: http://www.freepatentsonline.com/WO2018081920A1.pdf

[4] https://www.utsouthwestern.edu/labs/next-generation-sequencing-core/sequencing/

[5] https://patentimages.storage.googleapis.com/9d/a9/9c/697f0ed8d84cab/CN205133580U.pdf another patent, https://patentimages.storage.googleapis.com/8a/13/0b/727f18776a8f2e/CN205368331U.pdf also describes a 2 lane flowcell.

[6] https://patents.google.com/patent/CN205368376U

[7] https://patents.google.com/patent/CN206607236U

Following on from my previous article on the BGI and list of sequencing companies this post contains my notes on Complete Genomics (a BGI acquisition). If you have any further insights on Complete, I’d love to hear them. Please email at new at sgenomics dot org.

Business

Complete Genomics was founded in 2006 to develop a DNA sequencing platform based on a sequencing-by-hybridisation (SBH) approach. One of the founders (Dr Drmanac) has a long history of academic work in SBH going back to the 1980s. The commercial history of Drmanac’s SBH work also starts before Complete with a company called Callida Genomics  which was founded in 2001 as a subsidiary of HySeq.  There are Callida Genomics patents referring to methods currently used by Complete [6], which are now assigned to them. Drmanac also cofounded Hyseq, and it seems likely that some SBH work went on there. There’s therefore a commercial history behind the Complete Genomics approach extending back 10 to 15 years now.

Complete Genomics IPO’d in 2010. Crunchbase notes that Complete raised a total of 143M USD [12], and were valued at 232.2M USD at IPO. They were then acquired in 2013 by the BGI (for 117.6M USD).  The BGI seems to have transferred most technology development work to their Shenzhen site, cancelling new projects at the Complete Genomics Mountain View office [1] and making substantial staff cuts.

The BGI have however continued to develop the platform. Releasing sequencers for use in China under the BGISEQ brand. Competitively the Complete Genomics approach does not appear to have fared well against Illumina, but it remains and interesting technological approach.

Technology

Technology overview from [3].

The Complete Genomics chemistry, as described in their 2010 paper, starts with the formation of nanoballs of DNA (DNBs) [8]. They appear to have a neat chemistry which allows them to form nanoballs in solution which are not entangled with neighboring DNBs [7]. This is in contrast to other platforms which either require amplification to be performed on beads, and/or in droplets (emulsion PCR) or on a surface (Illumina clusters, polonys).

The DNBs are flowed onto a substrate (flowcell). This flowcell is patterned with an array of aminosilane features. The DNBs only bind to these features, resulting in a regular array. From what I can tell, only a single DNB can bind to each site. Without this they could have overlapping nanoballs (which would unusable), in general such systems are limited to about a third of site containing a single read (with multiple occupancy sites being unusable). Potentially this gives them a density advantage over other DNA sequencing platforms.

With the DNBs arrayed on the chip sequencing can begin.

[9] Chemistry overview from Revolocity document.

The image above gives an overview of the cPAL sequencing chemistry used in Complete Genomics instruments. This hybridisation/ligation sequencing process is probably my least favorite part of the Complete Genomics system. The process uses fluorescently labelled degenerate 9mers with a single known position [11]. So for example, to interrogate the first position the probes NNNNNNNNA NNNNNNNNT NNNNNNNNG and NNNNNNNNC might be used, each labeled with a different dye. After these are flowed in, ligated and imaged they are then removed and the next set of probes comes in. These would then interrogate the next position NNNNNNNAN etc.

I’d guess 9mers are used because the stability of shorter oligos isn’t good enough. My understanding is that they only label the first 5 positions. They then have a process for creating extended anchor probes “by ligation of two anchor probes allows decoding of positions 6–10 adjacent to the adaptor” [10]. This results in 10mer reads. They perform a number of 10mer reads at different adaptor sites and merge everything together. From memories of early datasets, reads have the potential for gaps because of this.

Un-captioned image from [3] Supplementary info. I assume the image on the left is the combined output of 4 images of a single cycle. Image on the right possibly represents crosstalk between dyes.

The process is complex and potentially error prone. However there is one possible advantage over the Illumina SBS approach. That is that each sequencing cycle in the cPAL system resets the template by removing the probes. In Illumina sequencing there is the potential for accumulated error (phasing error) as templates get out of sync (which ultimately limits read length). This does not exist here.

I’ve not looked at the original Complete Genomics raw data (I don’t believe any was released?). But I’d guess there is a potentially high raw read error rate (due to non-specific hybridisation among other things). It’s unlikely standard short read aligners would work well with Complete Genomics reads (being short and possibly containing gaps). For this, and other reasons Complete Genomics only ran a service business for many years. As I recall, they would only process human genomes, and delivered called SNPs to the customer rather than read data itself.

While the Complete Genomics systems were only used in house for a long time, since the acquisition of Complete by the BGI a line of commercial sequencers has been released under the BGISEQ brand. The BGISEQ-500 spec sheet suggests they are now generating 50bp reads [11] (the approach seems to be similar). Another instrument series, the MGISEQ has also been announced, which boasts a 100bp read length, however little information is available. It looks like fastq files may now be generated. Not much data seems to have made its way into the various public archives, but there is one report that suggests BGISEQ data looks pretty reasonable for SNP calling. Update: It’s not clear that MGI are pursuing an SBS approach, not SBH as used by Complete.

The approach is technologically interesting, but it’s difficult to see how it can complete directly with Illumina. However, it seems likely the BGISEQ instruments have a significant cost advantage at least in China.

Notes

[1] https://www.genomeweb.com/sequencing-technology/bgi-halts-revolocity-launch-cuts-complete-genomics-staff-part-strategic-shift

[2] Callida Genomics, Inc: http://www.evaluategroup.com/Universal/View.aspx?type=Story&id=13022

[3] http://science.sciencemag.org/content/327/5961/78

[4] Revolocity Video https://www.youtube.com/watch?v=WuS_RY8Zy38

[5] https://www.youtube.com/watch?v=DfaMOTcwcjs

[6] Callida Genomics Patent, referring to nanoball approach: https://patents.google.com/patent/US8440397

[7] “Short palindromes in the adaptors promote coiling of ssDNA concatamers via reversible intra‐molecular hybridization into compact ~300 nm DNBs, thereby avoiding entanglement with neighboring replicons” Science 2010 [3], supplementary info. Documentation for the now cancelled Revolocity states that “>95% occupancy of flow cell spots occupied by a single DNB”. http://www.completegenomics.com/documents/revolocity-tech-overview.pdf

[8] Using a controlled, synchronized synthesis, we obtained hundreds of tandem copies of the sequencing substrate in palindrome-promoted coils of single-stranded DNA, referred to as DNA nanoballs (DNBs).

[9] http://www.completegenomics.com/documents/revolocity-tech-overview.pdf

[10] The process is described well here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472021/

[11] https://www.bgi.com/us/wp-content/uploads/sites/2/2017/04/BGISEQ-500-ChIP-Service-Overview_linear.pdf

[12] Complete Genomics investors appear to include:

Enterprise Partners, OVP Venture Partners, Highland Capital Partners, Orbimed, Essex Woodlands Health, Prospect Venture Partners, Sands Capital Management, and ATEL Capital Group