Wednesday, June 24, 2015

Metagenomics and "A feeling for the organism"

Evelyn Keller's biography of Barbara McClintock is entitled "A feeling for the organism". A few paragraphs in this last chapter sum up quite nicely how McClintock viewed the scientific enterprise and discoveries:

"Over and over again, she tells us one must have the time to look, the patience to "hear what the material has to say to you," the openness to "let it come to you." Above all, one must have "a feeling for the organism." One must understand "how it grows, understand its parts, understand when something is going wrong with it. [An organism] isn't just a piece of plastic, it's something that is constantly being affected by the environment, constantly showing attributes or disabilities in its growth. You have to be aware of all of that.... You need to know those plants well enough so that if anything changes, ... you [can] look at the plant and right away you know what this damage you see is from-something that scraped across it or something that bit it or something that the wind did." You need to have a feeling for every individual plant. "No two plants are exactly alike. They're all different, and as a consequence, you have to know that difference," she explains. "I start with the seedling, and I don't want to leave it. I don't feel I really know the story ifI don't watch the plant all the way along. So I know every plant in the field. I know them intimately, and I find it a great pleasure to know them." This intimate knowledge, made possible by years of close association with the organism she studies, is a prerequisite for her extraordinary perspicacity. "I have learned so much about the com plant that when I see things, I can interpret [them] right away." Both literally and figuratively, her "feeling for the organism"

I agree wholeheartedly. Others may science differently, but I make my living by looking and studying everything about the bacteria I work with. Going into each experiment I have an idea of what to expect (even if these "experiments" simply involve streaking out cultures from frozen). If you give me a genome of Pseudomonas syringae, I can tell you the main components you'll find . I can tell you how certain strains will grow (or won't), what the colonies will look like, how long they'll take to pop up, what color they'll be. It took me a few years to gather this intuition, but now that it's engrained I like to think I have an innate sense when something is "off". I liken this to a scientific Spidey-sense. The challenging part is truly knowing when to follow up on these odd results, when to store away for the future, and when to disregard them as uninteresting.

I was reminded of McClintock's "feeling for the organism" by a couple of stories from metagenomics that have popped up across my feeds. Before I say anything else, I don't intend to denigrate the quality of the science or data underlying these stories by any means. The work is solid, I just think we're starting to find some limitations in the power of "big science" and these holes usually pop up in the discussion sections of papers and press releases. The first of these stories was a tour de force looking at metagenomics of the NYC subway system. The authors reported a variety of interesting results, but the tag line that a lot of news outlets seemed to focus on were the presence of Yersinia pestis (plague) and Bacillus anthracis (anthrax) within the subway system. The limitations of these methods have been hashed out already (here and here), but I want to focus on the inherent lack of "a feeling for the organism" when dealing with metagenomic data. Studies of any open microbial ecosystems are going to find a diversity of taxa. Unless you bring in specialists, there is simply no way to know the ins and outs of each organism. In the case of the NYC subway metagenome, from my interpretation at least, the authors looked at only bits and pieces of the Yersinia and Bacillus genomes without capturing the whole picture. They had to do this because the story was so inherently large that you couldn't possibly investigate everything in depth. However, specialists with "a feeling" for either Yersinia or Bacillus could have provided a viewpoint on which directions (other genes to look at, levels of nucleotide diversity which seem a bit too high) to follow up on to truly demonstrate presence of these bugs within the subway.

Likewise, one part of the story on urban microbes in this piece caught my eye:

"Rodents are under study, too. White-footed mice (Peromyscus leucopus) in New York City carry more Helicobacter and Atopobium bacteria — associated with stomach ulcers and bacterial vaginosis in humans — than their suburban counterparts"

I worked and slaved over Helicobacter pylori cultures all through grad school. I simultaneously loved and hated that bug. It's finicky growth patterns are the reason I moved over to study the reliably growing Pseudomonas after grad school. Unless something has dramatically changed since I've been in the literature (which is completely possible), rodents are terrible hosts for H. pylori strains that cause stomach ulcers (general overview here). You can get a subset of H. pylori strains to grow in mice, but there are often a variety of genetic changes that take place that allow them to adapt (see here).  I wouldn't be surprised if there were multiple Helicobacter strains within mice in NYC, but my money is on the fact that they aren't Helicobacter pylori that could cause stomach ulcers.

These are the tradeoffs that are made when dealing with immense data sets, and I'm not quite sure how to fix this. No one has a feeling for ALL THE MICROBZ. If you have a fun/interesting story on microbiomes that focuses on a couple of taxa in bold, at least try to run your data and ideas past someone that truly has a feeling for these organisms before publishing the paper. If it holds up after that, more power to you.

Friday, June 19, 2015

Navigating the waters of NSF grant submission

*Disclaimer: What follows is  a post about structural biases I've perceived within the NSF Biology system. I think these biases are intrinsic but keep in mind I could be completely wrong (and if you have different views, please feel free to comment). They also aren't inherently bad or need to be fixed, they just exist based on the pool of reviewers/panelists and timing of the grant cycles. It's a bit rambling, but I'm hoping to provide at least a slightly useful insight or two.

Even before the new office smell has worn off, and in many cases before you've actually moved into your office, the thoughts of many PIs newly merging onto the tenure track are focused on grant writing. This isn't going to be a post about how to get NSF grants, but more along the lines of "things I've experienced writing grants across panels". Grant writing is truly an art. Something that I didn't truly appreciate before is that, as with any piece of art, each target audience has their own subjective opinions. I've had my lab for 4 1/2 year now and have written grants to DEB, MCB, and IOS. I've been fortunate enough to sit on preproposal and full proposal panels. One of the most difficult ongoing lessons I'm learning is that grants written to each of these are very different beasts.

1) Preproposals change the game. DEB and IOS require preproposals, MCB does not. I'll save most of the comments about pre vs. full proposals for other posts, but suffice it to say that writing a convincing preproposal takes a different skill set than writing a convincing full proposal. Since preproposals don't go out for external review, the fate of your grant is entirely influenced by the composition of the panel. In panels that get a lot of submissions focused on similar systems (at least from what I've seen at IOS where there are only a handful of well-worn symbiosis models) novelty can be a benefit. If you propose to work with a new/novel system, and the science makes sense, you can get some bonus points if every other grant is focused on model organisms. Furthermore, while there are certainly benefits to working with a model system, it's more likely that someone on the preproposal panel will know little details about the nuances of the organism and can call you out for poor experimental design. On the other hand, if you are proposing to work in a system that no one on the panel truly has expertise in, you better be able to convince them in four pages that the experiments are feasible. Depending on overlap of the panel's expertise with your own grant, there could be details missed during the preproposal discussion/reviews, and their will likely be subtle misinterpretations. It's just how it goes and feeds into the noise of the system. These things can be ironed out in the full proposal though because those will go out for external review. I get the feeling that DEB grants and review panels have a much higher variance in topic and system than IOS panels. If such a difference truly exists it definitely adds a new psychological layer into the process.

One last thing to mention in regards to the effects of preproposals. There is likely to at least be a little overlap between reviewers of your successful preproposal and your full proposal. I can't speak to anyone else on this, but when discussing full proposals I remembered the discussions surrounding the preproposals. I remembered perceived weaknesses and strengths and I tried to see how the authors dealt with these criticisms. I can't help but think that it's a good idea to dedicate some of your full proposal to laying out a response to your preproposal reviews.

2) Timing can matter for CAREER grants, especially since you have a choice about which panel to submit to. Submission of IOS/DEB full proposals occurs in summer and overlaps with CAREER award deadlines. Panels evaluating full proposals for both of these programs will also evaluate CAREER awards at the same time. Given the vetting of ideas that occurs due to preproposals, differences between CAREER grants and full proposals were often pretty glaring. It's also possible that you could have turned your non-invited preproposal into a CAREER grant, and that it would be reviewed by the same panel for both IOS/DEB.

In contrast, the normal deadlines for MCB panels that I've applied to are now in November. Therefore, if I submit a CAREER award to MCB there is no chance that the grant could be reviewed by the same panel that it would be as a regular submission. This matters because I've had some CAREER grants go to what I perceive as weird places at MCB (like Engineering panels) and they get evaluated very differently than they do at the regular November panels. Differences in criteria between regular and CAREER grants aside, the science may be essentially the same in the grants I've submitted but I get a feeling that there is much more variance in the CAREER reviews simply because the panel isn't quite the fit I imagine it to be. I think this also factors in because I'm not convinced that reviews of CAREER grants inform my writing of regular MCB grants (and vice versa), whereas I think you can get more traction out of reviews regardless of grant type at both IOS and DEB.

3) Funding rates are low regardless, but DEB (evolutionary processes at least, I can't speak to anything ecology) feels like an even steeper climb for microbiologists than for other biologists. The first few times that I had grants rejected from DEB, the POs made statements like "you have to convince frog biologists that your work is important". These comments were spot on and looking back I did a terrible job at describing how my work applied across systems. However, and I could be wrong about this although the few people I've asked back up my intuition, I'm not sure that grants from frog biologists at DEB get the reverse critique of "convincing microbiologists that your work is important". I'm not sure what this means, and certainly some great microbiology work gets funded through DEB, but it feels like there is a slightly implicit bias from the reviewers against microbial evolution work at DEB. There are some generally important evolutionary phenomena in bacteria (like rampant horizontal gene transfer) that simply don't apply across systems. Likewise, there are some generally important evolutionary phenomena in eukaryotes (sex ratio biases, diploidy) that don't really cleanly apply to bacteria. Given the broad makeup of review panels at DEB, I think it's just hard to get some types of microbial work funded through there even though in a world with unlimited funding it's the right place for it. It's possible that the reverse is true at IOS because most model symbiosis systems involve microbes.

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