Can You Sequence a Bacteria’s Entire Genome Overnight?

Science postings here have been a bit light recently. I got a new job a bit back and it’s been keeping me pretty busy catching up on DNA stuff I haven’t really used since undergrad. Things are finally starting to settle down so I figure I’ll write a few posts about stuff I’ve been learning. So a lot of my job is helping to analyze the data from a shiny new DNA sequencer. Before I started, I didn’t know how far sequencing had improved in the last several years.

Until recently, most sequencing was done with Sanger sequencing. This type of sequencing produces about 100,000 bases per run and requires the DNA to be first grown in bacteria before sequencing. Then Margulies and a bunch of coauthors from a company called 454 published a paper in Nature and produced a commercial sequencer capable of sequencing 250 times as many bases per run. To do this, they used a technique called pyrosequencing. The process is pretty cool as shown in this figure from the paper.

Pyrosequencing bead preparation from Margulies et al 2005

The figure goes in a clockwise direction. On the top left, DNA is fragmented into many pieces. Next in the upper right, the DNA is bound to tiny beads, one piece to a bead and the beads are isolated in little bubbles where the attached DNA is copied millions of times. This leaves each bead with millions of copies of a single piece of DNA. Importantly all these DNA are single stranded and looking for a matching strand. In the bottom right, the beads are deposited one to a well in a fiber optic slide. Then helper immobilization and enzyme beads fill in the wells in the bottom left. You can see some real images of this process in their next figure.

Beads droplets and wells for pyrosequencing from Margulies et al 2005

The left photo shows one of the beads (thin arrow) in a droplet (thick arrow). The bead is about 1/30 mm in diameter and the droplet about 1/10 of a mm. On the right is a electron micrograph of the wells on the fiber optic slide where beads are trapped. Each well is about 1/20 mm wide.

Once all this has been setup, they get to the real pyrosequencing part. With all the beads firmly nested in their separate wells, the sequencing machine takes turns flowing the A, T, C and G nucleotide building blocks of DNA over the wells. Because the DNA bound to the bead is single stranded, these new nucleotides begin building the second strand nucleotide by nucleotide. The trick to this technique (and where its name comes from) is that when a nucleotide is incorporated pyrophospate is released. This pyrophosphate is converted to ATP (a very common energy storage molecule) by enzymes on the helper beads. The ATP then fuels a bioluminescent luciferase enzyme (like in fireflies) to produce light. A 16 megapixel camera captures this light and the number of nucleotides incorporated can be estimated from the brightness. By cycling through T, A, C, and G around 40 times, the machine can count the number of bases incorporated in each step and get an average read length of about 110 bases. You can see that process in the following figure with (a) the nucleotides ready to flow over (b) the wells with their beads and produce light which is captured by (c) the camera and analyzed.

Pyrosequencing machine from Margulies et al 2005

The authors were a little worried whether the shorter 110 base sequences would be useful. So they tried to sequence a bacteria, Mycoplasma genitalium. Although it’s sort of an easy target since this bacteria has a tiny 580,000 base genome, they did get an extremely thorough 40x coverage from a single run and were able to successfully assemble an accurate sequence of the genome.

The Rest of the Story

What they don’t mention in the paper is that one sequencer costs $500,000. Each run costs about $10,000 in chemicals and reagents (still cheaper than Sanger sequencing). Perhaps unsurprisingly at those prices, the 454 company responsible for this paper was later bought (for $150 million) by Roche, one of the largest pharmaceutical companies in the world.

Reminiscent of many gadgets, early adopters buying the sequencer from this paper got kind of screwed because 454 soon came out with a new improved model able to generate sequences twice as long. It looks like they’ll soon releasing an upgrade for the second model that should allow 2-4 times as many reads and again double the length (resulting in 8-16 times as many bases as this Nature paper).

The paper is a little short of pictures direct from the sequencing process so here’s a couple from a recent run. First, here’s an example of a single flow (a T nucleotide [no visible difference from other nucleotides]) showing 13 lanes of a 16 lane slide (you can divide the slide into portions to share the run [and the cost]). You might notice a pattern in some of the lanes. That’s because lanes 1-4 and 9-12 were tests to see how much DNA per bead produced the best results with the lowest concentration on the left.

Example of sequencing lanes from 454 pyrosequencer

And here’s a close up of a single lane during a flow (a C this time). Each bright dot signals incorporation of a C nucleotides. Brighter dots mean there were several C’s in a row.

Closeup of sequencing lane from 454 pyrosequencer

So a very cool technology. It’s pretty amazing that an entire bacterial genome (up to about 1.5 million bases [soon to be 6 million]) can be sequenced in one shot. Unfortunately, animals including humans have genomes of 2 billion or more bases so no one will be sequencing any individuals or endangered species without a few hundred thousand dollars to burn. But a little over ten years ago, you could get published in Science for sequencing the M. genitalium and here it was used as a simple test. It’ll be interesting to see where sequencing technology stands ten years from now.


Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., Bemben, L.A., Berka, J., Braverman, M.S., Chen, Y., Chen, Z., Dewell, S.B., Du, L., Fierro, J.M., Gomes, X.V., Godwin, B.C., He, W., Helgesen, S., Ho, C.H., Irzyk, G.P., Jando, S.C., Alenquer, M.L., Jarvie, T.P., Jirage, K.B., Kim, J., Knight, J.R., Lanza, J.R., Leamon, J.H., Lefkowitz, S.M., Lei, M., Li, J., Lohman, K.L., Lu, H., Makhijani, V.B., McDade, K.E., McKenna, M.P., Myers, E.W., Nickerson, E., Nobile, J.R., Plant, R., Puc, B.P., Ronan, M.T., Roth, G.T., Sarkis, G.J., Simons, J.F., Simpson, J.W., Srinivasan, M., Tartaro, K.R., Tomasz, A., Vogt, K.A., Volkmer, G.A., Wang, S.H., Wang, Y., Weiner, M.P., Yu, P., Begley, R.F., Rothberg, J.M. (2005). Genome sequencing in microfabricated high-density picolitre reactors. Nature DOI: 10.1038/nature03959


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Tangled Bank #104

Tangled Bank Icon Myers, Darwin and cake

Welcome to the 104th edition of the Tangled Bank blog carnival (a biweekly showcase of good biology posts selected by the authors themselves). Rigorous calculations and archaelogical research have revealed that this is the Tangled Bank’s 4th birthday. In the birthday spirit, several people sent appropriately themed presents.

Chris gave some great gifts (with a few caveats); a protien to resist radiation (may cause cancer), an enzyme to live longer, slimmer and stronger (but anti-socially and so far only in mice), and a transcription factor that can reverse skin aging (also in mice).

Flu virus

Even more microbiological gifts came in. steppen wolf wrapped up a nice box of cancer-fighting microRNA while Nimravid added some surprisingly robust bacterial gene networks. Finally, Ed chipped in a giant symbiotic bacteria with 40,000 copies of DNA and some influenza virus (straight from the flu’s tropical Asian source) and I contributed some cancer fighting bacteria.

I’m running out of synonyms for “give” and ways to twist submissions into presents, so let’s leave the birthday party behind and see the rest of the submissions.

First some plant related posts. Ocean Rambles has a bunch of nice pictures of the endangered Garry Oak ecosystem (and spring flowers) on Vancouver Island. Also concerned for plants, rENNISance woman links to the new idea of plant dignity (and a very odd stem cell comment thread).

On cultivated plants, Jeremy warns that relatively little money is being spent on farming research, especially for developing countries that need it most, and urges farmers to stop being pushed around by an agricultural corporation that sounds like the RIAA of farming (plus health effects).

Continuing the topic of corporate machinations, Biotunes describes an article (and personal experience) about bias in medical publications. On the lighter side of medicine, you can play doctor in space with a cool little flash game from the BBC.

Flagellum model

Moving on to scheming of the creationist sort, Greg theorizes why physics doesn’t have argumentum ad Nazium documentaries and points out that biology at the molecular scale is difficult to comprehend. Monado gives an example of this difficulty by comparing creationist drawings to a real electron micrograph of a flagellum. Late update: On the topic of pseudoscience, Podblack Cat asks “are women more superstitious?” (and throws in quite a literature review for the topic).

As an antidote to that intelligent design, Alvaro has details on making new neurons and a bunch of interviews of neuroscientists and cognitive psychologists.

Finally, 10,000 Birds (the only returning blog from Tangled Bank #1 [this post if you're curious]) describes coots (the bird, not the elderly).

I hope you enjoyed this Tangled Bank. The next edition is at the Beagle Project. You can email submissions to the hosts directly here or here or to the standard before May 14th. Here’s to four years of biology blogging carnivals and hopefully many more.


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Cancer Fighting Bacteria

I was doing a bit of background reading and came across an interesting paper about mutating normal bacteria into cancer-fighting bacteria. The paper centers around a single gene called inv (short for invasin) that can give an otherwise mild-mannered noninfectious bacteria the ability to invade cells.

Now this might seem like a pretty bad idea since there are probably enough infectious bacteria in the world already but this was only the first step of the research. Anderson and colleagues attached inv to a genetic switch (normally used for bacterial metabolism control) that turns on when arabinose (a type of sugar) is present. Unfortunately this switch was a little leaky. So even bacteria without arabinose were still infectious. Not ones to let that stop them, the researchers took out the ribosome (protein-making organelle) binding region of the gene, randomly mutated it and tested to find bacteria that were off by default but still able to turn on.

Once they got that working, they decided to attach a sensor to the infective gene. Bacteria often do things like switch metabolisms when they run out of oxygen. The researchers picked one of the bacteria genes that turns on when oxygen is low and replaced the arabinose switch from the previous bit with the oxygen sensing switch from this gene. Again the switch was leaky and they had to mutate it so it stayed off by default. Once that was done they had a bacteria that was only invasive in anaerobic environments. That’s pretty cool because tumors are often anaerobic (since they’re big lumps of fast growing dense tissue).

Plasmid for density dependent infectious bacteria

To go even further, the researchers tried to create bacteria that only turn on when there are many bacteria in one location. This will be useful because tumors often have higher concentrations of bacteria due to leaking nutrients and poor immune response. By creating a switch that only turns on when a bunch of bacteria are present, the bacteria can be further targeted to cancerous cells. To do this they used a gene from an ocean-dwelling bacteria that only turns on when many bacteria are present (the ocean bacteria uses the gene to detect when it has reached the light organ of squid). It seems odd that bacteria can communicate but it comes down to a simple mechanism made up of two genes. One gene encodes an enzyme that makes a chemical, called AI-1, that easily disperses in and out of the cell membrane. The second encodes a gene activator that is turned on by high concentrations of AI-1. When there are many bacteria, the environment becomes rich in AI-1 and the gene activator turns on even more production of AI-1 and gene activators. This positive feedback causes creates a sensitive switch that switches quickly from all off to all on when bacterial concentration crosses a certain level. By linking these genes to the infectious inv gene, the researchers created a bacteria that was only infectious when in high concentrations.

So now we have bacteria that might be able to selectively infect tumor cells. By combining this selective invasiveness with cell killing or immune response activating mechanisms, bacteria could become helpful tools for treating cancer (although there is still a pretty long way to go). The paper makes it look easy but that must have taken a good bit of work to get it all working so nicely. They ended up using DNA from three different bacteria species and many different bacterial systems. It’s always really cool to see how scientists can take DNA “parts” and combine them together to create new and useful functions and even edit the DNA directly when the parts don’t fit correctly.

I guess the next step in the research is to figure out how to get a bacteria to sense both an anaerobic and a high density environment. This might be a bit tricky since the two sensors would have to interact but I see some of the same researchers also have a paper on creating bacterial AND gates so I’ll have to give that one a read too.


Anderson, J., Clarke, E., Arkin, A., Voigt, C. (2006). Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria. Journal of Molecular Biology, 355(4), 619-627. DOI: 10.1016/j.jmb.2005.10.076


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Upcoming Tangled Bank

Tangled Bank BadgeIt looks like I’m the next host of the biology blog carnival Tangled Bank (if you’re not familiar see the last couple sessions for some good collections of biology reading). Pretty exciting since it’s my first hosting. Feel free to drop off any suggestions for stories in the comments here or email (I think those end up coming to me somehow).


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Gaza Strip Leatherback

There’s a new stories about a “rare giant sea turtle” being caught and eaten in the Gaza Strip going around now. I dug up a video for it and it’s definitely a leatherback so I figured I’d throw in my two cents since I just posted about leatherback turtles.

Here’s the video. Sorry if there’s an advertisement in front of it. I’m not making anything from it but it was the only source I could find. They do show the turtle being killed so it’s not really fun to watch.

A few corrections to the video first, it’s not “thought” to be a leatherback. There’s no mistaking a leatherback for any other turtle. That’s a leatherback. The fisherman says it’s 600-700 kilograms. I’m never good at estimating weights but the biggest one weighed in Canada so far was just a bit above 600 kilograms and that one looks on the small side so probably less than 400 kilograms (still a big animal though).

I thought it was odd they were talking about eating the meat since I’d often heard that leatherback flesh is poisonous but I can’t find a good citation for that and there do seem to be substinence fisheries for them so I guess leatherbacks are either edible or only occasionally poisonous. Also, I’d heard of people eating the eggs for ‘viagra’ effects but never the blood or the whole ailing children thing. I’m not sure what conditions are like in Gaza but if they’re not killing the turtle for necessary food, it really seem like a shame to kill an endangered species for bogus penis enhancement.

Also the fisherman says the turtle ruined a bunch of fishing gear. This is actually a common problem with leatherbacks. They don’t seem to have a way to reverse directions. So if they run into a net or even a loose rope in the water, they can easily become entangled. This can often end badly when the loops get caught around their neck and choke them or the tide rises while the ropes hold them underwater (or people decide to drag the turtle ashore and drink its blood to improve their sex lives).

In more encouraging news, the Reuters article ends with this:

A smaller Leatherback was caught off the Gaza coast last month but the turtle was released after fishermen discovered it carried a tag classifying it as an endangered species.

Thanks to William F. Landell for pointing this story out in the comments.


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