Russell's Blog

In my previous article, I outlined my plans for sequencing a very large number of metagenomes. Assuming that works, there also the problem of actually getting the samples in the first place. Aaron Darling likes to begin the story of metagenomics by saying, "It all begins with a spoon..."

So, how do you get the microbes from the spoon to the laboratory?

One of the things I learned from my experience in Kamchatka was just how tricky collecting samples in the field really is. From lining up permissions and paperwork, to dealing with cantankerous Customs officials, to avoiding getting mauled by bears, the trip from the spoon to the bench is fraught with difficulties. If you mess it up, you either don't get to do any science or you'll end up doing science on spoiled samples.

And then there is the DNA extraction. My lab mate Jenna published a paper last year where she created synthetic communities from cultured cells, and then examined how closely metagenomic sequencing reproduced that community. She found that the community representation was heavily skewed, but that the DNA extraction methodology was critically important. Because it was very difficult to know how well the extraction process was going to work on hot spring sediment, Albert Colman's group basically brought every DNA extraction kit they could lay hands on to Kamchatka. Also, they brought a whole lab with them; a 900-watt BioSpec bead beater (that almost killed our generator), a centrifuge, mini-fuge, a brace of pipetters, gloves, tips, tubes, tube racks, and a lab technician to run the show (see my Uzon Day Four post to see a little of that; also, most of the of heavy crates in the photos).

Albert, Bo and Sarah really did an excellent job pulling all of this together, but it was hard. Watching them (and helping them where I could) got me to think very carefully about how I want to conduct my field research. One thing is for sure; as much as I respect our BioSpec bead beater, I am not going to carry it into the field. Period. In fact, if I can possibly manage it, I am going to restrict my supplies and equipment to what I can carry in a daypack.

I'm still working on how I will do water sampling, but I think I might have found a solution to sediment sampling at the ASM meeting in New Orleans. Zymo Research just came out with a line of field DNA extraction kits that are intended specifically for field collection. The idea is pretty straight-forward; they combined a DNA stabilization buffer with a cell lysis buffer, and made a portable, battery-operated bead beater to go with it.

It's super cool, but I hemmed and hawed for a few months after ASM. I was a little suspicious of my own judgement; the system includes a cool gadget, and so of course I wanted it. I spent a month reading protocols and tinkering around before I finally decided that if the system works the way Zymo claims, it's just about the best thing for my purposes. What clinched it was re-reading Jenna's paper, which clearly shows the importance of thorough cell disruption.

So, I finally decided that I had to give it a try, and that's what this article is about. If you like, you can think of it as a parody of the tedious gadget reviews on Gizmodo and Engadget, with maybe a dollop or two of Anandtech's penchant for brain-liquefying detail.

I guess this wouldn't be proper gadget review unless I started with a meticulous series of photos documenting the unboxing. So, uh, here are the boxes.

The big one contains the sample processor, and the two smaller ones contain 50 DNA extraction mini-preps each. I'm going to leave the mini-prep kits sealed for now, since I'm going to use them for my field work. Zymo provides two DNA extraction mini-kits with the sample processor, so I'm going to use those to test out the system.

Underneath the documentation (directions are for suckers) and the mini-kits, there is the sample processor, a charging station, a 12 volt lithium ion battery pack, and an international power adapter. They also provide some little disks, which I think are for using with conical tubes (they recommend using skirted tubes, since conical tubes can shatter), and a couple of pairs of earplugs.

The earplugs turned out to be... prescient.

The sample processor itself is an modified Craftsman Hammerhead Auto Hammer. Upside? I can buy extra batteries from Sears! Downside? Seeing the $71.99 pricetag from Sears really makes Zymo's $900 pricetag hurt. Our super-powerful bench-top BioSpec bead beater is only about twice that.

When I asked, Zymo said that they've actually modified some of the internals of the Crafstman tool, but this might have just been to discourage me from traipsing off to the hardware store to buy some PVC pipe fittings and a hacksaw. Experience tells me, though, that I could easily fritter away $800 worth of time replicating their engineering. OK, $700. It's a really nice international power adapter.

I was a little disappointed to note that the Craftsman part is made in China. Not that I have anything against things being made in China, but I was under the impression that Craftsman was an American brand. It's a little like discovering that a jar of authentic-seeming salsa is made in New Jersey, or something. I'm sure they make perfectly good salsa in New Jersey. Nevertheless, I have a deep-seated belief that salsa should be made in a Southwestern state by grandmothers who each know five hundred thousand unique salsa recipes, and Craftsman tools should be made in Pennsylvania or West Virginia by guys who wear blue overalls and carry their lunches in pails.

OK, so maybe I do have something against everything being manufactured in China. While using the sample processor in the lab, it suddenly made a very loud click that I hadn't heard before. When I looked carefully, I noticed that there was a piece of metal debris caught in the motor vent. It seems to be made out of aluminum (it's not ferromagnetic). My guess is that this is debris from the manufacturing process, not a broken part of the device. I shook out two other smaller pieces, but lost them before I could photograph them. It looks like the three pieces are part of a square. Most likely this is the remains of an improperly handled punch-out, like a metal version of a paper chad. As you can see, it got kicked around inside the motor housing until it was ejected into the vent. I think Craftsman (or their subcontractor) should get the blame for this, rather than Zymo.

Here is the soil/fecal mini-kit. Each prep uses three sets of spin columns. The bead bashing tubes, as they are labeled, are in the upper right, along with two tubes of lysis/stabilization buffer and a tube of elution buffer.

The protocol says to add the sample first, and then add 750ml of lysis/stabilization buffer, and then bead-beat. But... then you would have to bring a p1000 and tips along with you. No thanks. The sample tubes and the beads had better be chemically stable, or they'd wreck everything. So, I aliquated the buffer into the bead tubes before leaving the lab, and left the p1000 behind. Zymo includes some very fancy spin columns with this kit; they have their own caps, and little nubs on the flowthrough channels that you need to snap off before you use the columns. I've not encountered anything quite like these.

The final step of the kit includes these green-capped columns that are pre-filled with buffer. I wasn't expecting any liquid to be in them, and so of course I spilled the first one on my foot. Don't do that.

So, I took a little miniature field expedition to the exotic environs of the Putah Creek Riparian Reserve to try this out. It didn't take long to find a place that promised to have plenty of microbes.

Here's a soil sample before processing.

I processed some of these samples for 45 seconds (the directions recommend a minimum of 30 seconds). Usually it seems to work fine, but occasionally the tube explodes and splatters mud and buffer all over the inside of the lysis chamber.

The exploding tube problem appears be caused by grit preventing the threads from closing correctly. In other words, it was my fault. Be extra careful to get the dirt actually inside the tube. Here's what it's supposed to look like.

After processing, the samples are noticeably warm. If you are going to process for much longer than 45 seconds, I suggest you stop and let the sample cool for a few minutes before continuing.

Here are the yields I measured for the mini-kit preps (minus the tube that exploded), eluted into 100 μL of buffer.

Source	Yield
Potted plant	80.6 μg/mL
River muck	0.669 μg/mL
River muck	1.13 μg/mL
River muck	0.595 μg/mL

I messed up the extraction protocol a little bit (and I used too much elution buffer at the end), but still got enough DNA to work with. Not too shabby for a first try.

I decided I had to throw these samples and DNA away because I don't actually have permission to use samples collected on UC Davis's campus. That's also why I'm not showing a gel.