Road to the $1 Genome

There’s been much talk of the $1000 genome. But it’s clear that the price will continue to drop even further. Ultra-cheap sequencing (and sample prep) would open up entirely new applications. The route to ultra-cheap sequencing may hold some surprises and it’s interesting to run the numbers. Let’s begin imaging our ideal sequencing platform.

DNA sequencers are often characterised in terms of throughput. That is, how much DNA they can sequence per unit of time. The human genome is about 3 billion basepairs. You’d be forgiven for thinking that you’d only ever want a sensor that delivers that much sequence. Turns out there are lots of applications where lots more sequencing would be useful. Often you’re sequencing larger populations of organisms. Or you’re looking for something that doesn’t happen very often (low abundance fragments of cancer in blood plasma).

As a convenient guesstimate I’d say we want to be able to sequence 1000x the size of a human genome. Oh and I’d like to be able to run this in 5 mins. For fear of being accused of overkill I’ll leave the specs at that (this much sequencing costs 1000s of dollars and would take days currently).

How many sensing elements and what kind of throughput would be needed? Sensing DNA at more than 100 bases per second (bps) is likely to be tough. Nanopore approaches generally generate signals in the picoamp range. Amplifying these signals at anything more than a few 10s of Kilohertz gets hard. SBS approaches will certainly also have issues running at speeds faster than this.

How many sensing elements do we need? 3^10^9/60/5 is 10 million. At a 100bps, that gives us 100,000 sensing elements.

An iPhone 6s camera can reliably sustain these kinds of data rates. 120fps at 1080p (~2megapixels). That camera module regularly goes for about $20 on eBay, similar modules are likely available OEM at much lower prices. Which is to say, we can build CMOS chips at volume, which produce data rates in the right ballpark.

While $20 is cheap, it’s still not cheap enough to be a throw away component in a $1 sequencing system. And while semiconductors are crazy cheap (you certainly can buy cheap CMOS sensors for around $1), it’s hard for me to imagine shipping a complete, semiconductor derived, consumable for this price.

So what does the technology behind a $1 run sequencer look like?

The great hope for the future of sequencing has always been nanopores. In this technique the DNA passes through a small aperture. As it moves through the hole the DNA sequence is read off using one technique or another.

Nanopore arrays are based around semiconductor fabrication, and the cost profile is likely to be similar. It’s hard for me to see how a nanopore sensor could be produced for less than $1, and delivered to a user for less than $10. It’s also hard for me to imagine that the array is reusable. Any system where the DNA is in contact with the sensor will result in the contamination of that sensor, and its rapid degeneration.

So what might a $1 sequencer look like? Somewhat controversially, I think it might decouple the sensing technology from the substrate in much the same way current massively parallel sequencing platforms do today. An optical system, with cheap reagents, and a cheaply manufactured substrate (which costs <$1) would seem logical (though a reusable FET sensor might work too). The instrument itself might use more expensive CMOS cameras and cost a few hundred dollars, but because the sensing and sample are decoupled they could be used repeatedly.

Disclosure: While I always try and remain unbiased. I own stock in sequencing companies. I’ve worked for DNA sequencing companies. And I continue to work in the industry. Exercise your own judgement.

Further Details on the Illumina Firefly

AGBT has come and gone, and with it further details of the Illumina Firefly platform have appeared. The speculation I previously posted appears to be largely correct. Genomeweb has an excellent report on what was presented by Jay Flatley. From the Genomeweb article we note “The Firefly device is essentially a CMOS sensor with nanowells. The nanowells are fabricated over photodiodes to enable DNA deposition to be aligned one-to-one with each photodiode. Clustering and sequencing then occurs directly on the CMOS chip.” and that Jay said it’s “inherently a one-channel device”. This means that they are using a single photodiode per well, unlike CrackerBio who have suggested that a triple-junction photodiode could give a decently specific signal dependent on differing wavelength.

A new chemistry has been developed for this one-channel system. He appears to have suggested that a dye needs to be cleaved and another added during imaging. That makes sense of course, but does add complexity to the chemistry. It likely means at least a slight decrease in read length/increase in error rate (all other things being equal, which of course they are not).

Using a single photodiode is the conservative move at this point, and makes sense to me when introducing an entirely new detection mechanism. Jay also said that they’ve demonstrated 150bp reads and error rates similar to the HiSeq X (to which I would say as always “show me the data”).

Announcements like this are interesting. Jay said it was because it would be difficult to keep it secret as they will be ramping up their supply chain. I’m not sure I buy that, they’re a public company and there’s always a pressure the break into new markets, release something new, and show share holders that things will get better and better.

Disclosure: While I generally try and remain unbiased. I have worked for multiple DNA sequencing companies, I continue to work in sequencing, I have interests in sequencing companies. As always, you should form your own opinions based on the facts available.

Personal Genomics Inc./CrackerBio

crackerbioI recently came across a company called CrackerBio. They’d not come up on my radar before so I did some googling. As always, it’s worth noting that I have interests in this area and as such you should take my notes with a pinch of salt.

CrackerBio was formed in 2007 [1], their early press releases indicate they entered the Archon X Prize in 2009 (the prize turned out to be a bit of a flop really).

They’re an optical-semiconductor platform, as such it’s easy to make comparisons with Firefly. They are however quite different approaches. For a start, the CrackerBio system is single molecule.

The company seems to have undergone something of a reboot in the last year and is now called “Personal Genomics Inc.”. There’s a great video from a recent conference from which much of this post is sourced. There are also a number of patents, which I have only skimmed at present.

Anyway, here’s an overview.  The prototype chip is shown below. They’re platform bares some similarity to PacBio’s. The incorporation of labelled bases on a strand sitting in a well is observed in real time. Unlike PacBio the whole system is integrated into a single chip.

crackerbio2

The CrackerBio Chip

The system looks quite neat, they plan to be to market in 2016. The CrackerBio site also showed 2015 as a launch date, and with semiconductor fabrication involved I’d guess there’s plenty of scope for delays.

Specs look quite reasonable, with estimates showing run throughput in the Gigabase range (much like Firefly). Unlike Firefly however they target long reads.

The system has a few neat aspects. A single molecule of DNA is attached to a bead, the beads sit in a nanowell. Under this well is a planar waveguide. My understanding is the waveguide is used to illuminate the bead. Planar waveguides in only one direction, so I guess this prevents the light from flooding into the sensor. The overall structure is shown below.

 

 

 

 

 

 

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CrackerBio Chip Structure

crackerbio5

Using the tri-gate photodiode for fluorescence detection.

The final piece that makes the system viable is the triple-junction photodiode. Photodiodes are in general pretty sensitive to light across the spectrum. When we use a CCD camera or other photodiode array we usually combine them with filters. Either a fixed filter in scientific applications or a Bayer filter integrated into the device as in typical consumer cameras.

In the CrackerBio chip concept I don’t think it would really be viable to integrate filters into the device. So they take a different approach. Rather than using a single photodiode, they use a structure that effectively gives them 3 photodiodes, each with different response characteristics. Any one photodiode would not give a response current which would allow the frequency of light coming from the fluorescent label to be determined accurately. However the hope is that by combining the 3 different signals (shown to the left) an accurate determination can be made.

Overall I think the approach looks pretty neat, however realtime observation of the polymerase at work has proved problematic for PacBio and resulted in high error rates. From the looks of the system they will have one read per sensor, this would limit throughput. Having to spin a chip to advance the platform, also brings its own issue (takes money and time). It’s an interesting concept however, and I plan to keep an eye on their progress.

 

[1] From here. ABOUT TEAM CRACKER -Initiated in 2007 with support from the Industrial Technology Research Institute (ITRI), team cracker is based in Hsinchu, the heart of the Taiwanese “Silicon Valley.” Under the direction of Dr. Chung-Fan Chiou, an engineer, innovator and business developer, team cracker blends the expertise of young, highly talented and innovative opto-electronics engineers, organic chemists and molecular biologists. The team is developing a new ultra-high speed, low cost and portable method to sequence long base-pair reads with high accuracy. It is based on the sequential conversion of photons to electrons and to DNA sequence, on a single composite chip. The versatility of the underlying design is expected to revolutionize studies of biomolecules at the single-molecule level. For more information, please visit www.crackerbio.com.

A Solexa Story

solexa_logo

whosolexa

Early promo shot from the old Solexa site. The 90s rocked!

In this post I describe non-technical aspects of the development of the Solexa sequencing platform. For those who don’t know, Solexa is the company that was acquired by Illumina. They developed (acquiring the necessary IP as required) a DNA sequencing platform several orders of magnitude faster and cheaper than what was previously available. The core chemistry developed at Solexa returns broadly similar to that currently used by Illumina, and which dominates the DNA sequencing market. If we decided to implement GATTACA today, you’d probably use Illumina machines.

Solexa are now long gone of course, the solexa.com now proudly boasts an exciting deep clean make up remover. Old versions are available via the waybackmachine.

solexanew

Kind of a shame Illumina didn’t keep the domain…

It’s interesting to look at how things played out financially. And what it cost to get to each milestone. In this post I’ve pulled some data out of their filings with companies house, and a SEC filing which describes their technological progress.

Briefly the following table describes the funding I’ve seen noted in the accounts and press releases (this maybe wrong and I’d welcome corrections):

raisetable

It’s instructive to plot this against certain milestones. These are available in a Solexa SEC filing.  You can see that they’d raised 34.4MUSD in total prior to sequencing anything at all. Getting the platform up and running, took time. The sequencing milestone is 9 months after they acquired the Manteia cluster amplification IP which proved critical. Things moved rapidly after this point.

raised

The total amount raised tells us something about how much it cost to develop the technology. It doesn’t however tell us about the valuation of the platform at each round. If you just plot the amount raised (rather than the total) you get the following:

raised2

The fact that the raise in 2004 was lower than the previous value makes me suspect that it could have been a down round? Based on the numbers calculated in the section below I don’t think so. The valuation possibly looked like this:

valuation

The interesting thing about this is that the valuation prior to a proof of concept looks like it was in the ~30->60MUSD range.  After getting sequence data the valuation must have increased rapidly. It feels like the acquisition price was quite low, and that it could have been single digit multiples of the last round valuation. So, those are my thoughts on Solexa’s valuation and fund raising, I will try and rework this and check/fix the calculations when I can. Below I detail some the background/sources and how I arrived at some of the numbers above.

Business Development Background and Sources

solexa_progress

Progress in data generation at Solexa from 1.

The company was incorporated in 1998 as “Intercede 1356 Limited”. As a UK based company all its prior accounts are a matter of public record. However you’ll now have to search under “Illumina Cambridge Limited” (the business unit Solexa became after acquisition).

Their initial funding came from Abingworth Bioventures II. The first set of accounts filed shows 300KGBP in cash and records a 300KGBP loss (it records transactions to the end of 1999). The company commenced activities on the 12 of October 1998. And I would expect this raise to have been around that point.

The year 2000 accounts are a little more explicit, and contain the following statement:

REVIEW OF THE BUSINESS AND FUTURE DEVELOPMENTS

The Company has been established to undertake research and put the results of such research to commercial use.

The Company raised a further £1.5 million during the year by way of a convertible loan from Abingworth Bioventures II SICAV (see note 8 to the accounts). This has been used to establish the Company in its own premises and to build the management and scientific teams. Good progress has been made in the development of the Company’s proprietary single molecule technology.

EVENTS SINCE THE BALANCE SHEET DATE

Subsequent to the year end the company raise £100,000 by way of a convertible loan from the University of Cambridge.

On 20 September 2001, the capital element of the above convertible loans of £1.6 million from Abingworth Bioventures II SICAV and University of Cambridge were converted into 800,000 ordinary shares of 0.25p each at £2 per share. In addition £4,104 of accrued interest on the University of Cambridge loan was converted into 2,052 shares at the same rate.

Also on 20 September 2001, the company raised £12,000,000 (before expenses) by the issue of 4,000,000 ‘A’ ordinary shares of 0.25p each at £3 per share.

So by my reading there was an initial investment of 600KGBP in 1999. Sometime in 2000 they got an additional 1.5MGBP from Abingworth. Then in 2001 an additional investment of 13.6MGBP. It’s also an interesting technical note that they describe themselves as a single-molecule sequencing company.

Going through the accounts the next interesting event is the acquisition of the Manteia IP:

On 25 March 2004, Solexa Ltd and Lynx Therapeutics Inc. jointly acquired from Manteia SA the rights to propriety technology assets for DNA colony generation. Solexa intends to use the Intellectual Property, in conjunction with its existing technology for the comprehensive and economical analysis of individual genomes.

This was the point that they transitioned from being a single molecule company to a cluster (or as they called it at the time colony) based sequencing platform. For those familiar with the technical aspects of the system this, is a significant change in direction for the company.

Prior to 2004 they were burning about 3 million a year it seems. In 2004 they were burning about 5 million (GBP). By my reading of the accounts they had about 5MGBP left in the bank, that’s after raising what looks like 8MGBP:

On 30 July 2004 the Company issued 4,166,666 ‘B’ preferred shares with an aggregate nominal value of £10,471 for gross proceeds of £7,500,00. On 18 October 2004 the Company issued a further 277,778 ‘B’ preferred shares with an aggregate nominal value of £694 for gross proceeds of £500,000.

That was probably raised from Amadeus (as noted in this press release), in fact the Amadeus press release notes 14.4MUSD which is almost exactly 8MGBP at that dates exchange rate. The press release also notes that in total Solexa had raised 40MUSD to date. The exchange rate in 2004 was almost 2 dollars to the pound, so this approximately matches the 22.2MGBP if you total up what I found in the accounts.

In March 2005 they were acquired by Lynx Therapeutics, Inc.. While structured as an acquisition, it was more like a merger (or Solexa acquiring Lynx) Lynx changed its name to Solexa, Inc continuing the Solexa brand. The deal was possibly largely done to give Solexa a US stock listing. I believe most of the tech development was still done by the Solexa guys in Cambridge.

Because of the merger, the UK accounts are less informative from this point on. But we can see that in 2005 they burnt 9MGBP. The accounts state they have 8MGBP left in the bank. And 2006 they appear to have burnt about 13.5MGBP. And in 2007 they were acquired by Illumina for what was widely reported as being 600MUSD.

Other sources of information help fill in the gaps. A press release from July 12 2005 contains the following:

Solexa Completes $24 Million Private Equity Financing

HAYWARD, Calif.–(BUSINESS WIRE)–July 12, 2005–Solexa, Inc. (NASDAQ:SLXA) today announced that it has completed a private equity placement for approximately $24 million following stockholder approval of the financing at the Annual Meeting of Stockholders held on July 7, 2005. The financing represented the second and final closing of the $32.5 million private equity placement that was announced on April 21, 2005. SG Cowen & Co., LLC served as the exclusive placement agent for the transaction.

“This financing demonstrates our investors’ confidence in our ability to execute on our business plan to develop and market our next-generation sequencing systems based on Sequencing-by-Synthesis (SBS) and Cluster molecular arrays,” said John West, Solexa’s chief executive officer. “In the coming months, as we move closer to product launch, we expect to be able to announce additional experimental results demonstrating the performance of our platform in high-end genetic applications.”

Under terms of the financing, the second closing included the sale of approximately 6.0 million shares of common stock at $4.00 per share and issuance of warrants to purchase up to approximately 3.0 million shares of common stock at an exercise price of $5.00 per share. The first closing of the private equity placement, completed April 25, 2005, generated proceeds of approximately $8.5 million from the sale of approximately 2.1 million shares of common stock and approximately 1.1 million warrants. As previously announced, Solexa’s prior venture capital investors Abingworth Management Limited, Amadeus Capital Partners Limited, Oxford Bioscience Partners and SV Life Sciences invested a total of approximately $10.8 million in the financing at the second closing.

Stockholders at the company’s annual meeting also approved all other items included in the company’s 2005 Proxy Statement. Among the measures were the election of seven nominees to serve on the Solexa board of directors for the ensuing year, including three affiliated with the company’s venture capital investors and one with ValueAct Capital, the lead investor in the private equity financing. Other approved proposals included adoption of the company’s 2005 Equity Incentive Plan.

Their early press release contains the same information:

HAYWARD, Calif.–(BUSINESS WIRE)–April 22, 2005–Solexa, Inc. (Nasdaq:SLXA) today announced that it has entered into a definitive agreement for a $32.5 million private sale of common stock and warrants for the purchase of common stock with a group of leading institutional investors in the health care sector. The transaction is led by ValueAct Capital. Solexa also announced that G. Mason Morfit, CFA, a partner of ValueAct Capital, has been appointed to its board of directors, bringing board membership to eight.

Under terms of the financing, Solexa will sell approximately 8.1 million shares of common stock at $4.00 per share and will issue warrants to purchase approximately 4.1 million shares of common stock at an exercise price of $5.00 per share. Approximately 2.1 million shares of common stock and approximately 1.1 million warrants will be issued in a closing expected on or about April 25, 2005, and the balance of approximately 6.0 million shares of common stock and warrants to purchase approximately 3.0 million shares of common stock will be issued on the same terms in a second closing subject to stockholder approval at the 2005 annual meeting. Solexa’s intended use of proceeds includes the development and launch of its first-generation Sequencing-by-Synthesis (SBS) molecular array platform for genetic analysis and repayment of its loan from Silicon Valley Bank. All of Solexa’s previous venture capital investors, including funds affiliated with Abingworth Management Limited, Amadeus Capital Partners Limited, Oxford Bioscience Partners and SV Life Sciences, will be investing a total of approximately $10.8 million in the financing at the second closing of the financing and have entered into agreements with Solexa to vote in favor of the financing at the 2005 annual meeting. SG Cowen & Co., LLC served as the exclusive placement agent for the transaction.

A press release from 11/21/05 contains the following statement:

Solexa Announces Agreement for $65 Million Private Placement

HAYWARD, Calif. and CAMBRIDGE, England–(BUSINESS WIRE)–Nov. 21, 2005–Solexa, Inc. (Nasdaq:SLXA) today announced that it has entered into a definitive agreement with a group of institutional investors to raise approximately $65 million from the private sale of common stock and warrants for the purchase of common stock. This financing will result in net proceeds to Solexa of approximately $61 million after deduction of offering expenses.

Under the terms of the financing, Solexa will sell 10.0 million shares of common stock at $6.50 per share and will issue warrants to purchase approximately 3.5 million shares of common stock at an exercise price of $7.50 per share. Approximately 3.9 million shares of common stock and approximately 1.3 million warrants will be issued in a closing expected on or about November 22, 2005, and the balance of approximately 6.1 million shares of common stock and warrants to purchase approximately 2.2 million shares of common stock will be issued on the same terms in a second closing subject to stockholder approval.

So that’s 97.5MUSD in 2005. I don’t see anything else prior to the acquisition. The final data (used for the table shown above is then):

1999 960,000USD (600,000GBP*1.6)

2001 19,040,000USD (13,600,000GBP*1.4)

2004 14,400,000MUSD (8,000,000GBP*1.8)

2005 97,000,000USD

2007 acquired for 600,000,000USD

It would be nice to get some idea of the valuation at various points. UK companies also publish share ownership. So we can roughly work this out. In the first filing in 1999 Abingworth owned exactly 50% of the company. For which they seem to have paid ~1MUSD. Valuing the company at 2MUSD. They received an additional 1.5MGBP from Abingworth sometime in 2000 increasing the stake to 60%. Valuing the company at up to 30MUSD (depending on how exactly these two investments were structured).

After this round a bunch of people jumped in. Amadeus, Oxford Bioscience, mRNA Fund II, Scroder Ventures, SITCO, and SV all invested in a 20.04MUSD round. The 2002 return shows that Abingworths stake dropped to 47%. I don’t think Abingworth put much in, so I’d guess the valuation here was around 50MUSD. After this round there was a round in 2004 which Amadeus led. If I’ve worked the numbers correctly then before/after that round ownership broke down as follows:

raise3

It looks to me like Amadeus put in basically all the cash. Other parties were largely diluted, though put in a small amount to help maintain stake. So of the 14.4MUSD I’m going to guess Amadeus put in 12MUSD. I should be able to work it more precisely but that ballpark, with a ballpark valuation for the company in the 30 to 60 million mark.