What the fungi do I do with my ITS library?

It’s been about 8 years since I started working on my first 16S rRNA PCR survey (of Drosophila gut microbes). At that time, I was occasionally asked, “what about Archaea or what about microbial Eukayrotes?” Then, and ever since, my reply has been that it’s hard enough to get a handle on what’s going on with the bacteria – I don’t need to make my life more challenging by broadening my scope.

But, finally, this month, I’m making my life more challenging. As part of my new Seagrass Microbiome Project, I’ve decided to tackle the fungi. As far as I can tell, ITS is the “barcoding” marker of choice for fungal types. For many reasons, it’s best to follow the herd when doing this sort of thing: 1) someone else has already designed, tested, and published results with these primers, 2) there is a reasonably large database of ITS sequences available to compare my sequences to, and 3) I lack the interest and personnel to explore an alternative approach.

So, I just plunged right in. At first, I tried some new primers designed by Nick Bokulich, but he warned me that they were “finicky” and he was correct. I got no amplification with my seagrass samples, and the positive control I had only worked about half the time. I know some other fungi people, well, I know Jason Stajich (@hyphaltip), so I asked him which primers I should use, and I decided to go with the primers set used in a cool paper by Noah Fierer’s lab, in which they looked at fungi in rooftop gardens in New York City.

Those worked, and a few days ago I got word that my sequencing run was in. It looks like crap. We typically get about 12 million sequences from our MiSeq runs, but this time, I only got 4 million. I was also told that the reverse reads looked much better than the forward reads.

So, now, in addition to working with a new “barcode,” I have to troubleshoot a crappy sequencing run. In many ways, it’s nice to have undergrads and a technician in the lab who do all of my lab work for me these days, but it sucks when it’s time to troubleshoot because I’m so far removed from the bench that I have no idea what’s going on anymore.

So, the first thing I asked for was the Bioanalyzer trace that’s always run before the library goes on the machine. It looks like this:

Bioanalyzer trace for my first fungal ITS MiSeq run

Bioanalyzer trace for my first fungal ITS MiSeq run

I had been told that there was size variation. I had even seen some of the PCR gels. But, still this is not what I expected to see. Upon seeing this, I am concerned about two things. 1) If there is strong preferential amplification of smaller DNA molecules during the bridge PCR on the flow cell, then will I even see DNA from those larger peaks? 2) With our 300bp reads, for sure the amplicons in the peaks <400 will have overlapping forward and reverse reads, but for sure the 676bp amplicons will not. What effect will these two things have on my analysis? How do I accommodate this size variation? One of the reasons to follow the herd with these methods is that other people have probably already encountered and dealt with exactly this issue, so I turned to Twitter…

There are some great resources suggested here. I know what I’ll be reading this weekend…

MicroBioBlitz

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As soon as I was introduced to the concept of a BioBlitz, I immediately emailed the boss man Jonathan Eisen saying that we should arm these people with microbial sampling kits. He, apparently having received the same BioBlitz announcement that I did, was seconds away from sending the same email to me!

We contacted Jack Gilbert at the Earth Microbiome Project and Janet Jansson at LBL/JGI/JBEI, and George Phillips, the organizer of the Mount Diablo BioBlitz, and they were all on board to help make a MicroBioBlitz happen.

A few days ago, I received another announcement about the 2nd annual UC Davis SEEDS BioBlitz. So, yesterday, I got the go-ahead from those organizers, last night I threw together 20 swab kits, and today I took them to the Putah Creek Riparian Reserve for a test run of the MicroBioBlitz concept. It was So. Much. Fun! The BioBlitzers were super-excited to start swabbing their organisms of choice. I gave them no guidance, and I can’t wait to see what they decided to swab! Before I left, they had swabbed lizard butts and oak galls, and they were on the lookout for otter scat!

I couldn’t stay for the whole event because Vivian needed a lunch and a nap, but before we left, I had her swab a Valley Oak for us! She loved helping, but cried when I took the swab away from her, so I gave her a swab to play with, and she spent the next 20 minutes swabbing everything she could reach!

MBL #3 – The Volta Experiment

Before you even have a chance to really get to know your classmates, you are led on a dark walk to a stinky Cedar Pond, where the brave venture chest-deep into the cold, sulfuric muck. They stomp around in the soft, slimy, decomposing leaf litter and sediment, releasing bubbles of methane gas. The methane is trapped by one person (Dan Buckley, in this video) in a stopped funnel. On the count of three, Dan removes the stopper and Steve Zinder holds a flame as the methane shoots out of the funnel. Voila! Volta!

MBL # 2 – Jorg Overmann and my pet phototrophs

I mentioned in my last post that one of the many wonderful things about the Microbial Diversity Course at the MBL is the quality of the invited speakers. Another favorite of mine was Jorg Overmann, who wrote the book on Molecular Symbioses and is the director of the DSMZ (I did not know that!) He introduced us to his favorite bugs, the consortium known as Chlorochromatium aggregatum.

A really nice summary of what we know about them is here on the Small Things Considered blog. Basically, there is a flagellated Beta-Proteobacterial rod that is encrusted with non-motile green sulfur photosynthetic symbionts (epibionts, to be precise, because they are attached to the surface.) Their relationship is complex, but what’s overtly cool about it is that the rod can chauffeur the epibionts around in search of the right conditions for photosynthesis. One can (and we did) see their cool scotophobic behavior in a light microscope. It looks like this (run towards the light!):

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Jorg said that I should be able to find these consortia in the nearby Trunk River, so hello, of course I will try to enrich for them! However, he strongly discouraged my reliance on their enrichment for my independent research project in the last half of the course, because they are quite elusive and grow very slowly.

So, let me backup a little here. The first three weeks of the course you attend lectures in the morning and then you are in the lab until late at night. Those weeks are spent attempting to culture a diversity of isolates from environmental samples that you collected in the first few days. You are also introduced to all of the super-cool equipment housed at the lab, to which you will have full access. For the last three weeks of the course, you draw upon your new-found knowledge, the support of those around you (students, instructors, TAs), and the abundant resources at the MBL to work on an independent research project. Most people work on AN independent research project, but I apparently am not most people. I worked on FOUR. This is the first (and favorite) that I will describe in a series of blog posts.

Setting up the enrichment is easy. You just take some sediment from the river, put it in a bottle with some spring water supplemented with sodium sulfide, making sure that there is no head space and no way for oxygen to get in. Then, incubate it at room temperature with a light-dark cycle and wait.

Then you wait. I waited for 23 days. Some of them had purplish top layers of sediment and some had greenish top layers. Jorg said that Chlorochromatium aggregatum will form a biofilm on the surface of the glass. A biofilm formed! On the surface of the glass! It was purple.

Then you play with your biofilm. First of all, before I proceed, I did NOT find any consortia, but I hope you will not be too disappointed, because what the hell was I going to do with them in the week that I had left anyway? My biofilm consisted primarily of two cell types (see below.) You can see big, oval cells that contain highly refractive sulfur granules, and you can see some smaller, dark rod-shaped things.

I decided to use one of our cool toys to see if I could physically isolate the presumed purple sulfur bacteria (encircled in yellow) from those other guys. I wanted to isolate them 1) to get a 16S PCR product to identify them and 2) to see if I could transfer them from the biofilm into new culture medium and grow them.

I used the PALM CombiSystem which is a laser dissection/optical tweezer type microscope. Basically, I could spread out my biofilm on a slide covered by a very thin membrane, then use a laser to cut out a circle around one or more cells on the membrane, and then use some mysterious quantum force to catapult the membrane+cell(s) directly into the cap of an Eppendorf tube (filled with culture medium or whatever.) What the… what!?!? That was supercrazycool! Somewhere I have a movie of it, but you can check this out to get an idea:

So, I was able to isolate these guys (they were Chromatiaceae, per RDP) for 16S PCR and I was able to get them to survive the transit into the Eppendorf tube. After two days, they were still metabolically active!

These figures are from my final report. I must admit that I wrote the bulk of it, while drinking beer, and during an all-nighter. So, I wont vouch for it’s quality and can’t recommend that you read it. These reports, regardless of the quality, are good to have around because current students pore over old reports for inspiration. Reading it myself after 3 years, I felt a strange combination of “Wow, I did that?” and “Eww, I wrote that?” But, my goal here is just to share a little bit of my MBL experience with the world, and I hope you enjoy it.

If you were at Lake Arrowhead, you should know the MBL

The recent Lake Arrowhead Microbial Genomics Conference (#LAMG12) was the first time I’ve been reunited with one of my classmates, Ben Tully, (@phantomBugs) from the MBL. (In June 2009, I spent 6 weeks at the Microbial Diversity Course at the Marine Biological Laboratory in Woods Hole.) On several occasions, we explained to people at the conference how we met, and were met with blank stares.

Just as Laura Sauder (@LauraASauder) forgave the medical microbiology folks in the room for not recognizing a photo of Sergei Winogradsky, I am willing to do the same for those who do not recognize the Microbial Diversity course at the MBL. But, if your flavor of microbiology has anything to do with the words environment, community, genomics, metagenomics, evolution, or ecology, then you should fall into one of three categories: 1) You went to the course. 2) You are going to the course. 3) You want to go to the course.

Here is some text from the course website:

Students will isolate, cultivate, and experiment with characteristic microbial types from various marine, freshwater and terrestrial habitats, including those microbes living symbiotically with animals and plants. Emphasis will be on the isolation and cultivation of organisms that are distinguished by their phylogenetic, physiological, and morphological properties. Techniques for cultivation of strict anaerobes and phototrophs will be emphasized. Examples of microbial types that will be isolated are methanogens, acetogens, sulfate-reducing anaerobes, fermentative anaerobes and both oxygenic and anoxygenic phototrophs, as well as bacteria involved in the geochemical cycling of various metals. Magnetic bacteria, sulfur-oxidizing bacteria, spirochetes, and luminescent bacteria will also be studied. A laboratory component on molecular approaches to microbial diversity will instruct students to use approaches of molecular phylogeny and comparative genomics. This will involve isolation and amplification of 16S rRNA genes as phylogenetic markers and the use of computer software programs to analyze nucleic acid sequences and to construct phylogenetic trees. As the capstone activity of the course, participants will conduct an individual research project of their own design.

You will learn a LOT about microbes and how they do what they do. You will become a microbiologist. The way you view the world will be permanently altered. Just ask anyone who has attended the course. But, what that blurb does not tell you is that every day, you sit through lectures or talks by the world’s expert on a given subject. Time to learn about thermophiles? Here is Karl Stetter. And, not only will he talk to you in the classroom about his research, he will stick around for a few days. He will hang out in the lab, eat meals with you, and he will be part of the crowd that makes the trek to the secret beach when the bar closes.

It’s just a really, really special place, and if you were at Lake Arrowhead, if you were at ASM, if you were at ISME, (I know there are more, but you get the idea) you must fall into one of those three categories.

More MBL posts to come…

Mechanical Turk

Yesterday morning, my last day at the Lake Arrowhead Microbial Genomics Conference, I saw a tweet from Holly Bik (@Dr_Bik) about a talk she was attending at a Phenomics conference about the sociology of Amazon’s Mechanical Turk Web Service. What is Mechanical Turk, you may ask? Well, what’s really funny is that just minutes before I had answered that question for Ben Tully (@phantomBugs) in describing how I used it to help with my dissertation research.

Mechanical Turk allows you to crowd source little tasks that are easy for humans, but not computers. For example, if you need to write a short caption for 1000 photos at about 1 minute per photo, that would take you about 2 full days of work. Or, you can upload those photos to Mechanical Turk, along with some instructions about how to write each caption. Each photo becomes a little job, sent out to all the workers on Mechanical Turk. You offer to pay $.03 per job, and then you sit back with a glass of wine and watch the World Series of Poker. Some hours later, all of your work is done, and you did none of it. Sure, you are out $30, but hopefully your time is worth more than $15/day.

You are provided with the worker ID for each job. You can spot-check each worker’s work and if you do not like it, you can reject all of their work, do not pay them, and then those photos go back into the work queue

So, how did I use this for my dissertation research? I wanted to look at environmental correlates of horizontal gene transfer (HGT). HGT is the exchange of DNA between different species, and it is fairly common among microbes. One potentially important mechanism of DNA transfer is the uptake by a cell of DNA that is floating about freely in the environment (transformation). If the environment is inhospitable to the DNA molecule, then the probability of transfer by transformation should be quite low. For example, and in particular, I wanted to ask whether organisms that live in very low pH environments experience a lower incidence of HGT.

I can predict the incidence of HGT for an organism directly from the genome sequence, so all I need is to find out at what pH that organism grows. That should be straightforward because every time a genome is submitted to a public database, the submitter will include all of the associated environmental data (or metadata) that is available, and since that submitter grew the organism in culture in the laboratory, he or she must know at which pH it best grows. Right?

Now, because I am who I am, I want to do this analysis in a phylogenetic context, using some Phylogenetic Comparative Method (I should talk about this more in another post.) In particular, I opted to use Felsenstein’s Independent Contrast method as implemented in Phylocom. I built a reference phylogeny for ~800 bacteria and archaea which have genome sequences available (this part is currently in revision) and then I “looked up” the optimum growth pH for each of them. This look up process should be straightforward, too, because the data are submitted to a searchable database, like at NCBI or the JGI’s IMG. Right?

Well, nothing that should be straightforward ever is in my academic life. I was able to get the pH data from the IMG by grabbing all of the webages with the metadata on them and parsing them with a little perl script. But when I did that, I only got pH data for ~100 of the ~800 organisms in my reference phylogeny. For any given organism, if I spent a couple of minutes poking around in the literature, I could easily find the optimum growth pH, so it’s not like it’s not out there. But, I couldn’t automate the “poking around” process, and after spending two full days of work, I had only retrieved pH data for about 200 additional organisms. Because I was down to the wire in terms of a dissertation submission deadline, and because I consider my time fairly valuable, I just couldn’t bring myself to keep at it.

I was complaining vociferously about those ~600 people who couldn’t be bothered to spend their couple of minutes to include pH data with their genome submissions. Russell Neches (@ryneches), who seemed to really get a kick out of my uncharacteristic vociferousness, suggested that Mechanical Turk might work for me.

I created a job (called a HIT): “Given the name of an organism, report it’s optimum growth pH”

My job looked like this:

In retrospect, maybe I could have come up with some better instructions, but I think for most things, these should work. I paid $.08 per job, so I spent $40. I rejected a lot of work because I got answers like: 37.5 or comments like “I couldn’t find it.” There was one worker who commented, “This was a cool HIT.” And that worker did a lot of jobs and seemed to do good work. I focused my spot-checking on values at the extremes, since most things live at a neutral pH.

But, here’s what I found: If you follow the first approach that I suggest and google the organism name+optimum+pH+growth, sometimes, you would see something that looked right in the google search results, but was actually referring to enzyme activity rather than growth conditions.

I can tell that this is not the information I’m looking for, but I don’t really expect a Mechanical Turk worker to be so discerning, especially not for $.08. There were a few other common errors that I think it would have been difficult to avoid, even with more clear instructions. I could have submitted some of the jobs in duplicate so that I could check the workers against each other, but I suspect that this type of mistake would be made by anyone without a fair degree of specialized knowledge on the subject (who is not likely to be doing this sort of work.)

So, in the end, I ended up spending a lot of time double-checking the results. I don’t know if I saved any time by doing it this way, but it was FUN! I will definitely keep it in the back of my mind, and hope to be able to use it again someday!

Catch-up

UPDATE: After I wrote the post below, I stopped posting to this blog for several years. During that time, I got married, converted to Judaism (although it still feels weird to call myself a Jew, mostly because my husband is not so observant – he did not ask me to convert, by the way –  and I am agnostic), had a baby, finished my PhD, and started working as a Project Scientist in Jonathan Eisen’s lab. Most of my previous posts were about hiking and a road trip that I took from Davis to Durham to attend a workshop on Phyloinformatics at NESCent. During that road trip, I stopped along the way to collect Drosophila because I thought that the Drosophila-microbiome interaction was going to be the main focus of my dissertation research. One of my strokes of genius was to solicit the help of big-name Drosophila biologists at regular intervals along the way to help me out. I asked them to take me collecting with them when I got to their town. My plan was to camp all the way from Davis to Durham and back. Without fail, everyone I contacted either offered to take me out collecting, put me up for the night, spent an entire day with me, identifying and sorting species in their lab, or all of the above. It was an awesome experience that I really should write a book about.

Anyway, I’ve finished the PhD and feel like this new chapter in my life should be accompanied by a renewed dedication to this blog. So here we are.

 

Post proper:

I’ve been a slacker about this, so I’m going to try to slowly catch up to the present. On my way home from Angel Island in April, my car hit a milestone. Here it is:

I didn’t know at the time, but this marks the beginning of the era in my car’s life in which most everything falls apart. I guess you could think of it as a time for a re-commitment to my car. Time to re-invest the purchase price.

Buying a 1992 used VW Cabriolet: $2000.
Keeping the car running for the next 100,000 miles: well over $2000 (I’m afraid to add it all up.)
Getting out of Davis whenever I want to: priceless