Life Science Tools of the Trade

…we’ve also installed one of these:

Yes, that’s a Roche GS-FLX, which will be fully loaded with their latest “Titanium” upgrade. Actually, it might already have it – I confess I’d have to ask someone in the lab, or maybe the office next door. Joining the other instruments from Illumina and Applied Biosystems, and promising greater than 400-base paired-end reads, this should solve a lot of (biological) problems. We hope.

But – are we behind the curve, again? Pacific Biosciences has announced that they are gearing up for an early-access program in the first half of 2009 (and I apologize if you can’t see that article, which might be subscription-only), meaning that they might actually have functional instruments on the market in 2010 as they’ve been saying since February. If this thing actually works as advertised, it will be game-changing in the extreme, and will make the other three competing instruments look stodgy, expensive, and slow.

PacBio has thoughtfully provided a nice little video on their website for you, if you’re interested in this technology. Warning – the term “zero-mode waveguide” is used, although the rest of the description is very basic. The claim is made that entire genomes (or, presumably, genome-sized complex template mixtures, like transcriptomes) will be sequenced in under an hour, at a cost of hundreds of dollars. If true, this will just about spell the end of the microarray for most applications.

We’ll see, I guess. Applied Biosystems certainly has other, competing technologies in the pipeline, but I doubt very much that Illumina has the resources, or Roche has the vision, to stay competitive in the long run. As for Helicos, well, I’ve changed my previous optimistic tone and have switched to predicting their demise for a while now, although they recently bravely reported that they will sell five to ten instruments by the end of the year. I’m not convinced, but I guess we’ll see. In the meantime, the Illumina, Roche and AB instruments appear to be working now, and our three-pronged, high-throughput attack on biology is continuing unabated.

Interesting times.

One of the pipette companies had a *mini* vendor show here the other day. As everyone probably knows, I don’t think very highly of pipette calibration companies. This company, however, also sells different brands of new pipettes as well as refurbished ones.

Since we have way too many pipettes in the lab to replace them all with new ones, I’m thinking about getting some refurbished ones. The price is a lot less than new pipettes and the refurbished pipettes have a 90 day guarantee.

I figure I’ll try one new set and see how it goes.

I’ve spent considerable amounts of time here, elsewhere on the Science Advisory Board, and in a few other places, writing about so-called “next-generation” sequencing. While some vendors are already shying away from this “next-generation” moniker, it’s a good enough catch-all phrase for now, I think. Anyway, most people seem to know what it means.

The currently available technologies include the Illumina (née Solexa) Genome Analyzer II (replacing the flaky Genome Analyzer), the Roche (née 454) GS-FLX (replacing the original GS20), and the Applied Biosystems SOLiD (still in its initial release). There’s also the Helicos HeliScope, which as far as I can tell is kind of like the mythical white elephant, or the Loch Ness Monster – many agree that it exists, but nobody’s ever seen one.*

Jumping in to this game rather cautiously, we ran a very unreliable GA for a while, and after considerable beating up on it have obtained some very nice data (in particular, for some ChIP-seq experiments, where the miniscule 30-ish base reads from this instrument are ideal). It’s now in the process of being replaced by the GAII, and for the time being both are co-existing on the same bench.

Spot the difference.

But, not to fall prey to the single technology trap, we’ve also added an Applied Biosystems SOLiD, which is currently being installed. Here it is with its doors open (look away, children).

This sequencer photo is rated PG-13 for partial nudity.

And, just to make sure that our bases are completely covered, we’re also ordering a GS-FLX, since it seems there will be quite a lot of genome-sequence backfilling in our future, and the long reads (with reliable paired-end capability) should do the trick.

Fun and games. Our old 3730xl capillary sequencers would be feeling sad, if they weren’t so darned busy screening for mutations and making sure everybody’s plasmids are what they’re supposed to be.

A few years ago, this NGS stuff all seemed like voodoo. Now it’s here, and, like any good scientists, we’re itching to play with it. Good times.

*I have shamelessly stolen this analogy from a colleague. It can equally well be applied to the results of certain experiments that are supposed to have been done around here.

The lab budget and the books are my boss’s job. I just try to keep the lab stocked and the supplies ordered for the lab. That said, I shop around a bit and buy when items are on sale. Most of our lab supplies have stayed pretty constant through the years. Restriction enzymes, tissue culture supplies, chemicals, the prices on these *old* lab items haven’t changed much and they’re not that expensive.

What I am noticing is the expensive price of all the *new* supplies. The RT-PCR reagents, the deep sequencing supplies, the modified oligos, FACS analysis… those items are pricey! The control for the RT is a couple hundred. The oligos a few hundred. Is it the new technology or the times dictating the prices? I’m not sure. It does make me wonder though how some labs can afford to do these experiments. I’m assuming the price will eventually come down on these items. In the mean time, I wonder how many experiments aren’t performed because the cost of doing them is just too high for the lab.

In this post-90’s biotech era which we all inhabit, I’m finding that brand recognition is becoming a confusing game indeed. Old favourite boutique vendors like Molecular Probes have been swallowed up by enormous, multi-tentacled distributors like Invitrogen, and keeping track of who’s distributing your favourite brand of pipettor, or water filter, or tissue culture media, can make for hours of fun and games. Even the big players keep getting bought and sold – just try to sort out the whole Merck/EMD fine chemical business, or the ownership structure of VWR, if you have some time to kill. And I’m still trying to get my head around Thermo Fisher. What does Thermo Electron have to do with distributing pipette tips and latex gloves? How, if at all, is this related to Thermo Finnigan? It makes my head hurt just thinking about it.

And then there’s my personal favourite suite of technologies du jour, loosely grouped into “next-generation” DNA sequencing, or NGS. Illumina buys Solexa, Applied Biosystems buys Agencourt Personal Genomics (but not Agenourt Bioscience, which is owned by Beckman Coulter – are you following this?), Roche gobbles up 454 Life Sciences. Pacific Biosciences is next, mark my words, with rumours of intense interest from Applied Biosystems, and probably many others. Helicos too, perhaps, so look for a merger or acquisition there, although with a market cap of $147 million and $50 million in the bank at the end of 2007, they could probably stay on their own for a while.

In the case of Illumina’s almost-works-most-of-the-time Genome Analyzer, most people still call it a “Solexa”. At the recent AGBT conference, which I’ve rattled on about in more detail elsewhere, practically the only people using the term “Illumina Genome Analyzer” were members of the large posse of Illumina employees in attendance. And most people don’t call the ex-454 machine a “Roche” GS-FLX; to most, it’s still a “454”, although this seems to me to be waning a bit under the crushing weight of Roche’s marketing machinery. Remarkably, the 454 Life Sciences website still exists, and is still a much, much better source of information on this NGS system than the Roche website, which is large, messy, and rather full of the 150,000 other things that Roche sells.

On the other hand, Applied Biosystems seems to have triumphed in branding their SOLiD system, and virtually nobody seems to remember that this was developed by Agencourt Personal Genomics and that the chemistry used was, for a time, referred to as the “APG process”, even by AB itself. Now it’s just SOLiD, small “i” and all, and the scientific community in general seems to have accepted that brand. Timing, I suppose, is everything.

Now, if someone could just explain to me why all these darn NGS boxes are blue…

A co-worker asked if I’d help her with some cloning. She had the strategy all figured out. The oligos were here and the pcr was done for the insert. All I had to do was digest the insert fragment and the vector backbone, purify them and ligate them together. Seemed like it should be easy enough.

That was until I looked at the enzymes. Urgh! They were two of those enzymes you only use under dire conditions. One cuts at 37 degrees, the other at 50. AscI only cuts 100% in NEB buffer 4, the other one only in buffer 3. I gave her a hard time about passing the more *difficult* cloning on to me. She said she knew about the temperature difference but that both enzymes cut in buffer 4. Huh? I had just checked the NEB catalog and knew that wasn’t right. BsaI only says it has 50% activity in buffer 4.

What was going on? We both felt like were going crazy. She brings her 2005-2006 NEB catalog over and shows me. BsaI and AscI both cut in buffer 4 – 100%. I look in my 2007-2008 NEB catalog and BsaI only cuts 50% in buffer 4. Turns out were both right. Sorta.

I’m not sure what changed. Is NEB purifying the enzyme differently? Did they just realize it didn’t cut as well as they had thought? Whatever the reason, the recommend buffer has changed. I’m not sure how many other buffers have changed but if you have an old NEB catalog hanging around you might what to get a newer one!

PS – Turns out neither one of us has had a whole lot of success with the cloning so far. I’m still not sure which is the best buffer to use!

It just arrived! DHL just delivered the free t-shirt that I signed up for a couple of weeks ago. It came shrink wrapped in the shape of a t-shirt. Do you know what I mean? Those shirts you can buy that are packaged to look like different shapes. I’ve seen shirts and towels being sold like that but never got one. Holy Cow! You open up the package and the shirt is so wrinkled. It really is amazing.

Promega gets credit for following through on the shirt promotion. I have to admit, though. I’m not sure I’ll wear a shirt that says “Research makes me do it.”

Okay.  I didn’t think about it.  I guess nobody did.  Since when do you have to specify inside delivery on an order?
The other week, I ordered a fair bit of pipet tips.  Turns out, the order turned out to be a pallet worth of tips.  The company shipped them freight from California.  Probably cheaper shipping costs for them and it doesn’t matter to us how they’re shipped.  What does matter to us was that they were delivered to the lab.  They were dumped on the loading dock.
The truck driver actually came up to the fourth floor to inform us that he didn’t have orders to make an inside delivery.  All he had to do was leave them at the loading dock.  Okay.  Not thrilled about it but not a big deal.  We could go down and get them.  The irritating part of all this was when he offered to deliver them upstairs.  With the use of our cart and for an additional fee, he’d bring the order upstairs.   WHAT?!?

I still can’t figure it out.  Was this a way for him to make a little extra money?
Needless to say.  We didn’t pay him to bring the tips upstairs.  Three of us went down with a cart, pulled the pallet apart and *delivered* the tips the lab.  One cart, one trip, no extra delivery cost.

By now everyone should know our lab uses Operon (www.operon.com) for all of our oligos.  When I say oligos, I mean TONS of oligos.  We generally receive at least one order a day from Operon.

That said, when I was ordering my oligos today I noticed the money in our account was getting low.  I submitted the required paperwork to add funds to the account and before the day was out – our account had been credited.  Now, that didn’t really impress me.  What impressed me was the email I received soon after.  Jeremy, our Operon rep, sent me a thank you note for the order.  It was just a quick note saying thanks and if he can help with anything to give him a call.

Little gestures like that go a long way in our lab.  We have the most respect for sales reps that know what we order from them and say thanks for the order.  It’s a small simple  gesture that goes a long way!

Wotcha folks, long time no see.

I’m going to cheat here, and link to a piece I’ve just written elsewhere, because it fits well under Tools of the Trade.

The word alone chills the hearts of experienced cell biologists. And when, a couple of months back, someone upstairs was getting strange results from their cell-based assays, the Boss came to me and asked,
‘What do you know about mycoplasma?’. The icy black hand of dread gripped me and I spent the rest of the day trying to find a supplier for a mycoplasma detection kit in Australia.

Please forgive me for this blatant self-plagiarism.

Ahh, the joys of the Holidays. Nobody is around, its quiet, and you think you are going to get some work done. But I think it is too quiet, and therefore I search the internet for amusement. Oh well, at least I’m working on digesting all the food that was force fed to me over the long weekend.

One quick update. Since the hbogerd’s last posting the Qiagen tech support has called the lab again. Evidently they had his email wrong, and wanted to shoot him a email. I guess they are still reading the blog. I’m sure we’ll get an interesting update when the masked man returns from his holiday respite. Personally, I think Beta testing would be great….if there was some incentive. Hbogerd has already made mention of the reasons not to, so the incentives should be pretty nice. Just a thought.

Alright then, how ’bout a little contest. Reply to the post with your most outrageous company/lab holiday party stories, and when one is left that beats mine, I’ll post that for your amusement.

Finally, after months and months of hearing nothing about the Qiagen fuzz problem, the rep informed me yesterday that yes, there was indeed a problem with their kits.
In case you missed the initial complaints, everyone in our lab was having problems with fuzz in their DNA maxi preps. After numerous phone calls to tech support and little if any resolution, we gave up and changed to Roche DNA kits. Qiagen didn’t seem to concerned with the problem and they certainly did little to appease us. (After spending tens of thousands of dollars with them…you might think they would want out business.) Apparently, the fact that the lab has done over thousands of maxi preps wasn’t enough to convince them that we did in fact know how to follow a protocol. Instead, it was somehow our fault we were getting fuzz. The tech support phone calls were infuriating to say the least and we had had enough!!
All of that being said….the Qiagen rep stopped by yesterday and informed me that it wasn’t us and there was a problem with their kits. FINALLY! After six months they agree with us that their was a problem. Something to do with the kits sitting on loading docks in extremely high heat. I’m not really sure of all of the details and I really don’t care but I do find it very disturbing that a company takes that long to recognize and identify a problem.
We have completely switched to Roche Genopure kits (I highly recommend them) and haven’t had any problems thus far. My advice to you…if you’re having problems with a Qiagen product….switch to a better company!

This is on-topic, honest.

A well-known crisp company (that’s ‘chips’ to you colonials) ran an offer last month. Every bag of crisps had a unique code stamped on it that got you one entry in an hourly (hourly!) draw to win an iPod. One an hour. For a month. That’s an imperial lorry load of iPods.

Needles to say, I didn’t win (boo) but I suddenly find that I’m inundated with offers of free or ‘chances to win’ iPods of one flavour or other. Let’s take a look at the week to date’s haul of junk mail – three items, so not too bad.

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What reminded me of the farce I went through in my previous job was a mailing from Flowgen, who are distributing (in the UK, at least) Gentra’s VERSAGENE™ range of DNA purification kits – genomic DNA that is, from blood.

Superficially it looks like a method that was developed at the company I worked for about 7 years ago. The methodology was based on a particular filter paper from a well-known filter paper manufacturer that had special properties. The manufacturer had a line of forensic papers that it supplied to police forces around the world, and we were particularly interested in DNA profiling (we had a state of the art ABI machine and could do SNP analysis, fingerprinting, the works).

So we had this method that involved 1 ml whole blood, two solutions, a column, a heating block and 10 minutes. Whole blood was applied to the column (which was basically a holder for this filter paper), sucked through under vacuum, lysis/binding solution added, suck, wash with same, suck, add water/TE and elute with heat. This last stage I seem to remember did not require negative pressure; the eluate just dripped through. And all this happened in under ten minutes. Yeah, the DNA was shagged – single stranded and 20 kB max – but it gave beautiful PCR data. Probably because it was single stranded.

It was a world-beater, but marketing were not happy. Oh no – my brief was to scale this up to handle 10 ml whole blood, using exactly the same technology (and also apply the same technology to bacterial cultures for plasmids, yeast, faeces . . . – I kid you not. That’s the kind of mentality I had to deal with), handling twenty four (24! That’s a whole unit of blood!) samples simultaneously. I played around with it for a week and announced that it could not be done. It wasn’t possible to elute the DNA from the filter in columns that could handle 10 ml blood without cooking it completely. It could be done if the protocol could be extended to an hour (the time it took to filter the solutions was much longer than for the 1 ml prep – this is obvious if you know anything about vacuum pressures and cross-sectional area), but even then yields were pretty poor. Nope, marketing wanted 10 minutes and no black pudding.

I wasted six months of my life on the project before landing a job I saw advertised in Nature and was replaced by two people who still hadn’t got the 10 ml scale prep working two years later.

But the point is the bloody management insisted that we stuck with that particular filter paper, despite me clamouring that we needed to try something else. I swear the CEO was on the take. Had we been able to find a more suitable matrix we’d have had a decent product, without an RBC lysis step and five solutions, five years before Gentra came out with this kit which – let’s be frank – is early 1990s technology at best. And this is symptomatic of the industry – there has been no real innovation in DNA extraction technology in ten, fifteen years. Yet Qiagen, Promega, the rest of them are slapping us with 800% markup on each and every prep they sell.

You’ll forgive me if I’m somewhat disillusioned.

Of course, it’s not just the science that requires reliable suppliers of quality equipment and consumables. The scientists, too, need victualization. I once read on Usenet that the four food groups are sugar, salt, fat, and chocolate. Caffeine and alcohol, however, are among the essential vitamins (Steve VanDevender).

Of these nutrients, scientists generally can not do without at least two, and usually three, of them on a weekly or more frequent basis. Free pizzas at trade shows and grad student soirees satisfy the fat and salt requirements, but this phenomenon is not widespread outwith the continental US. Caffeine, on the other paw, is consumed widely with much ceremony and gratefulness the world over on an incident timescale of minutes to hours. Alcohol is frequently used as a broad-spectrum analgesic and muscle relaxant, and when diluted with water is also very useful for cleaning and sterilizing benches. On average, we are looking at a timescale of hours to days here. Ultimately, the scientific world runs on various incarnations of the humble cocoa bean, and that my lab mangler does not like it is further proof of her continuing contribution to the oxygen deficit.
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I helped a guy in our lab spec a new iMac at the beginning of August, and he put the order in a few days after I went on holiday.

It’s only just arrived.

I’ve ordered a top of the range G5 and 23-inch flat screen so that I can solve the solution structure of the protein I wrote about last week. Order went in on Monday. Any bets as to when it’ll turn up? And I’m out of here at the end of the year. ARGH.

Apple – great products, lousy company. I guess it’s part of the mystique: You’ve got to really, really want one before they’ll ship. They’re probably running background checks on me as I type – “Is this person good enough for a Mac?”.

After three weeks’ sweat I finally got my protein sample, labelled for NMR, 99% pure at 10 mM in the fridge. It’s been difficult, the student couldn’t get the thing to express in minimal media so I was brought in to try, and after an epic battle which involved suspect TEV protease, chloramphenicol-S-Transferase and finally doing things the old-fashioned way, I got there. It goes on the magnet on Monday.

But what I came here to talk about is the supplier of the 13C-glucose and 15N-ammonium chloride we use to label the protein. Spectra Stable Isotopes are the guys, and they offer a not unreasonable price tag with their reagents. 13C-glucose, for example, is ~US$140 per gramme, so my experiment came in at about 600 quid, which is a bargain really (especially when you consider that I ended up with 150 mg protein at the end. What am I going to do with it all?). How do they do it?

I’ve often wondered about making these labelled compounds (and they do amino acids and suchlike, too). Organic synthesis? Euch.
Well, apparently they can synthesize complex molecules, but for most of the catalogue they let Algy do the work. Not Algy, algae. They feed isotope-enriched salts and carbon dioxide to algae, shine the sun on them and let the little blighters do all that boring intermediary metabolism stuff, then smash them apart and extract labelled goodies.

Clever, eh?

Our Institute changed tip suppliers over the summer. As you might imagine, with 400 scientists it’s not a small contract. The lucky people (STARLAB) who won the contract to supply our Stores don’t distribute the best tips on the planet, but I don’t really care since The Powers That Be wouldn’t stock the low-retention plastic ones I’d like in a month of Sundays. If I want them I’ll order them myself, as it would be a bit expensive to have them as our routine supply.

While the trials were ongoing (you think we don’t trial them before Admin make a cost-based decision anyway? Shame on you), the STARLAB reps picked up on the fact that many of the people who tried their tips were complaining that they kept falling off their pipettes. So when they landed the deal they came in and said right, we’ll calibrate and service all your pipettes (Gilsons and others) and replace the barrels if necessary. For free.

That was nice, and they steamed through hundreds of the things in jig time. I don’t know how thorough the service was, but my pipettes got new ‘O’ rings and seals, and the calibration certificate is pretty.

Of course, the day I got my Gilsons back from the STARLAB guys I got (yet another piece of) junk mail from Anachem, offering to service my Gilsons at a reduced price. Ha. Thing is, the service from Anachem is hardly cost-effective; after three years it’s cheaper to dump the old ones and get replacements, rather than having them serviced. Someone needs to wake up to this.
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It must be “Bash Qiagen Week”. Makes a change from “Bash Microsoft Century” I guess.

Chap in the lab has a (free, I assume. There’s no other reason to wear it) T-shirt from Qiagen that boasts the motto “Innovation in Bioseparation”. And I can only assume it’s not talking about the big Q because they haven’t innovated in fifteen years or more.

Let’s see, when I was working at a DNA separation technology company I revamped their kit product line. One of the improvements I made to the miniprep kit we sold was to include ‘Eppendorf’ tubes with extra long linkers between the tube and the lid. This was so that users could cap the tube with the filter bucket in place, obviating the need to cut the lid off when eluting the DNA at the end of the prep: maybe not a big change but it addressed something that annoyed the goolies out of me when performing Q minipreps, not least because I tend to write on the lids of eppies rather than the sides.
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I’m thinking particularly of K, who worked for BioRad when I was in Oxford. K was one of those rare (at the time) creatures who had a doctorate to go with their company car. Naturally she used to try to sell us BioRad gear, but she’d never lie to us if she thought another company did something better.

She understood the need to keep customers happy, and allowed us to trial a low pressure chromatography set up before the Department let me buy one, and naturally when I left that place I told all my friends (what’s that? Both of them? Yeah, hah bloody hah) about BioRad kit and tried to get them to buy it. K also didn’t wear strategic grade perfume and was very good at building rapport with the punters. I seem to remember going out to lunch down the Botley Road and – on K’s instigation – putting condoms over the end of the boss’s car exhaust. She went into the gents to get them, so I couldn’t really refuse.

I don’t remember why she left BioRad, probably getting ticked off at the way they treated her (and her customers). She went into business with some friends, selling stuff as a small, independent distributor. Got some nice things from them, including my favourite Nichimate stepper pipette and some very good low retention pipette tips (not coated; the beauty of these was that they were made from low retention plastic). She’s since moved on again, and I’m not sure where to. I should drop her an email and find out.

What companies seem to fail to grok is that reps are how people see the company. You get a good rep, one the punters like and get along with and can trust, and you get a good reputation. Reps like these, companies need to do all they can to keep them. And they don’t. Unfortunately the company and the punter lose out as a result.

Mood: green
Random musical fact: the opening chord of two riffs then an interval of a flattened fifth in Jimi Hendrix’s Purple Haze was a musical device condemned by the Spanish Inquisition.