Dammit Jim!

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.

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.

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.

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.

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.

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.

References

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

We thought it would be nice to have an easy simple wedding. Luckily we’re in Pennsylvania where, thanks to an historical abundance of Quakers, they have self-uniting weddings. Unlike normal wedding which require a priest or official, self-uniting weddings only need two witnesses. It sounded kind of cool to do our own wedding, so we gave it a shot.

So we went to the local Orphan’s Court (a nice pleasant sounding name) office to get our license. Things were going smoothly but once we asked for a self-uniting license, the clerk immediately asked us if we were Quakers and, when we said we were not, refused to let us register. Now I’m no legal scholar but when a government official asks your religion and then denies you service based on your answer, it sure sounds like somebody is mixing their church with their state. This was especially annoying because I had already read up on things and knew that the ACLU and a couple who wanted a self-uniting marriage had already brought the case to court and won. But I’m no lawyer and Philadelphia’s (more sensible) court house is just as close so we just went there the next day. The downtown Philadelphia clerks didn’t hassle us (although they are strangely very strict about having some piece of paper with your social security number on it) and we paid the extra $10 for a self-uniting license and got our license a few days later.

So, if you’re getting married in Pennsylvania and want to do your own ceremony, a self-uniting license might be slightly more hassle but should be pretty easy to get either by calling the ACLU or going to a non-backwater clerk (e.g. not Delaware County). A self-uniting marriage might also be a good option for people who want a friend or relative to marry them since Pennsylvania is currently invalidating marriages by ministers it doesn’t like (again I’m no lawyer but how does that sound at all constitutional?).

I just had a nice simple wedding a couple weeks ago (that and the new job is keeping posts even more sparse than usual). We weren’t doing anything big but I thought it’d be cool to dress up, especially since Xiaofen already picked up a wedding dress in China. So I started looking around for tux rentals. Surprisingly (to my uncultured self at least), tuxedos cost $150 to rent and you need to give them several weeks notice. This seemed a bit high so I went home and looked around the internet for typical prices. It turns out tuxedos really do tend to cost $150 to rent. But while googling, I noticed that at least one online store sells complete tuxedo packages for around $300.

Now I don’t mind spending money when I have to but when somebody wants to charge me 50% of the purchase price to borrow something for the day I’m going to draw the line. And get a bit annoyed. So I thought I might as well write it up here and maybe help someone else avoid getting screwed by tuxedo rentals.

I was a little worried there would be some sort of trick to a tuxedo that (relatively) cheap but the tuxedo arrived two days after I ordered it (I’m having amazing luck with shipping recently) and the package really seems to cover everything you need (except shoes and socks); jacket, pants, shirt, vest, bow tie, handkerchief (apparently called a pocket square) and simple cuff and button links. I did need to get the pants hemmed but I guess that’s normal and it was only $8 at a local dry cleaner. I’m no tuxedo expert but it seems to look pretty sharp and feels nice and comfortable.

So if you need a tux, buy4lesstuxedo worked well for me.

Details only interesting to people looking for tuxes: I thought the Geoffrey Beene tuxes looked the best out of all of them (although I wonder how much of that is due to the photography and model choice). I wanted a shawl or peaked collar (I never would have predicted I’d be writing about tuxedo collars on here) but they had a pretty low selection of both in my size. So I ended up getting the 2 button notch collar by Geoffrey Beene.

I couldn’t find any non advertisement pictures on Google so if here is a picture of a Geoffrey Beene 2 button notch tuxedo (and a beautiful bride) for any tuxedo shoppers.

I’ve got an upcoming (simple, very low-key) wedding and managed to put off getting a ring until the last moment (shoppings for jewelry is not really on my top 10 list). So I had pretty simple requirements; 1) silvery (which, since platinum is too expensive and you have to baby silver, I thought meant white gold), 2) affordable (over $400 really started me thinking about how much better other toys or travel sounded), 3) some sort of pattern or carving so it wasn’t just a boring band and 4) available within a couple weeks (me procrastinate?). After looking at about ten or so local jewelry shops, I was starting to get pretty depressed. Most of the rings were boring or really didn’t seem to look right (e.g. diamonds) and the few that I took a shine to either would take a month or more to resize/order or were way outside of what I felt like paying. Once I started running into the same rings in different shops (always suspiciously identically 50% off), I decided it was time to see what the internet had to offer.

After a bunch of googling, the online shops also seemed pretty generic and boring (although a bit cheaper). Then I started noticing the “alternative” metal rings. Apparently, rings made of silverish titanium or tungsten are gaining in popularity. Titanium is harder, lighter and a lot cheaper than gold and tungsten is apparently unscratchably hard (but shatterable) and a bit cheaper than gold. Having a ring made out of a cool metal instead of sissy precious metals sounded pretty cool (although maybe just a touch of classy metals might be good). But again a lot of the rings seemed sort of boring and/or expensive. The tungsten rings especially seem to have few choices and none with any good patterns or carving (I guess because they’re so hard to cut).

Then I found Boone Rings. Apparently a guy down in Georgia used to make titanium bike parts but decided to take his knowledge of titanium (and CNC machine) and switch to making rings. From his website, you can pick from about a hundred ring styles and then pick what metals to include in the ring, what curvature and width to make the ring and what sort of polish to finish the ring with. The idea of designing my own ring sounded pretty cool and a few of the various polishes seemed to scratch my itch for a pattern on the ring. So many choices led to analysis paralysis but I finally ended up deciding on a peened ring with palladium (pretty cool sounding fancy silverish metal) and white gold inlays. I also ordered a smaller peened white gold inlay one for the fiancee since she’s always worried about messing up her slightly fancy engagement ring and we thought it’d be kind of cool to have matching rings. Amazingly they arrived at my house 4 days later.

They sure look good. I like how the inlays are sort of hidden in the peened pattern.

I wasn’t sure what the ring widths looked like compared to fingers and I couldn’t get google to give me any good image of various widths on hands so for any similar googlers here is what 3mm ring and a 7mm ring look on fingers.

So if you’re looking for good looking (and extremely fast) custom rings with great customer service, I recommend Boone Rings.

I like to monitor my comments by RSS. That keeps all the fun blog related stuff in the feed reader and leaves my email for important stuff. Unfortunately, this has resulted in some comments languishing unnoticed in the moderation queue since there doesn’t seem to be an easy way (that I found with a quick search) to get the comments awaiting moderation into a feed. So here is a very simple plugin to put comments awaiting moderation into an RSS feed.

Features

• RSS feed of comments awaiting moderation

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