Funding versus Science

Yesterday, I ended up doing none of the things I intended to do, but cleaned them instead: (1) No bench-work, but a clean bench (and fridge and freezer spaces)!; (2) No emails, but a clean inbox!; (3) No thinking, but a clean mind! (Well, moreso than usual.)

My ears were also itching, because I realized that the NIH panel that's review my postdoc grant application was meeting, and was extra-worried, since now the Genome BC grant almost certainly will depend on the outcome of those scores.

Anyways, towards the end of the day, I switched over to browsing journals, which I rarely do these days...

I found a lot of good stuff I probably already should've known about, but I also found this opinion piece in PLoS about "what's wrong with funding of research". Pretty much sounds about right. I am not even in a position to have the stresses described in the article, but feel like if I don't get some kind of postdoc fellowship of my own, I'll be that much less likely to get hired as an independent researcher, since so much of what defines success is the ability to get money. But all of the PIs I know are overwhelmed by exactly these issues. It's especially daunting to realize that a full-sized NIH R01 grant can barely support a lab with 2-3 people.

On the other hand, my recent experiences with Rosie do bring home the fact that grant-writing is a good way to think rigorously about one's plans, and the Genome BC experience in particular (whether the grant is funded or not) helped our brains wrap around exactly what we'll be doing in the next couple of months. (continued...)

Decompression

Whew! Rosie and I just made what I consider a heroic effort to produce a grant application to Genome BC to use DNA sequencing to measure recombination biases during H. influenzae natural transformation.

It was heroic not only because we finally decided to apply only late last week, but because our co-funding support is tenuous at best. Genome BC requires that we match their funds at least equally with funds from another source. Rosie has funding, but it was applied for too long ago and the proposal only indirectly relates to our planned sequencing. We also applied for a CIHR grant recently, but will not have reviews until after the Genome BC committee meets in early January. The best hope of adequate co-funding comes from my NIH postdoctoral fellowship grant application (a resubmission) late this summer, for which I should have scores (or lack thereof) within a couple of weeks. If the application gets a good score, we can tell Genome BC that the major threat to the success of our application is ameliorated.

Almost immediately after submitting the grant application with Rosie last night, I had to turn to editing my buddy's manuscript (which I am an author on), which takes on the weighty topic of detecting rearrangements in complex mammalian genomes from limited sequencing data. I just turned my edits over to the corresponding author, and now, after letting the excess nitrogen out of my bloodstream, I need to decide what to do next.

Since aforementioned buddy also has taken several DNA samples off my hands for sequencing, I think I'd best turn to purifying uptake DNA from the periplasm of competent cells. I've already gotten things fairly well under way (see here, here, and here), but there's a bunch of uptake experiments waiting to be done. So tonight, I'll inoculate some cultures, so I can try a large-scale periplasmic DNA prep tomorrow, and tomorrow I'll also order some more radiolabel for doing more sensitive uptake experiments. (continued...)

Searching for Cash (How I'll spend my summer vacation)

So it’s time to outline my plans for the next several weeks... Grant-writing and preliminary data gathering!

Two grant applications to do:
(1) Michael Smith Foundation for Health Research (due June 25 to Office of Research)
(2) National Institutes of Health (due August 8)

I’ll find out whether or not to submit a full application to (1) in the next several days (if my “letter of intent” was sufficiently cool-sounding, I guess). The proposal itself is short (only 3 pages), so I should be able to focus on having a well-written piece in the next couple of weeks. This will help me a lot with the other proposal too. (I’d also better solicit letters for that one soon, but I want to wait until they tell me to apply first!)

And for (2) much editing, writing, and analysis to do! Rosie and I had begun tackling my rejected NIH proposal after I first got here, but enough time has passed that we’re both out-of-the-loop on our own editing. There are several things to do, besides polish the writing:

First off, the basics of the proposal are these: (a) I want to feed genomic DNA from one Haemophilus influenzae strain to a competent cell culture of another strain. (b) I’ll purify the donor DNA from various cellular compartments (representing different stages in the natural transformation pathway). (c) Then, I’ll use sequencing to measure the relative abundance of different DNA sequences along the pathway. (d) This should give me a comprehensive view of transformation potential of one genome into another.

There are a huge number of possible analyses, each of which may or may not be interesting. One major issue is how to present the kinds of analysis I’d plan on doing (and showing the reviewers that I can do it). And how to show that it really is an important set of experiments that I am capable of doing.

Proposal writing: Alongside simply improving the writing, I need to specify more details on how I will conduct the data analysis and provide any form of preliminary data that I can. These will also help us with the larger DNA uptake proposals we plan to submit in the Fall.

Background: I need to make the importance of the research plan and the specific questions I’ll answer much more clearly written and accessible. I also need to better understand the history of uptake signal sequences and how they were discovered. I.e. what’s already known versus what I’m going to learn. Rosie and I have talked this section through pretty well. It just needs to get re-written now.

Preliminary analysis: In my first version, this was just more background, but since I'll have been here a few months by the time I resubmit, might as well show what I've been doing...

1. I need a more direct comparison of the donor and recipient genomes I’ll be using. I’d previously culled data from this paper, but showing that I can do the comparison between the genomes myself will likely go a long way with the reviewers. I’ve got some pre-preliminary analyses here in this blog, but I'm still feretting around with that data and still learning how the alignment algorithms work.
   
2. I want to demonstrate that I can purify donor DNA from the periplasm and/or cytosol, so that reviewers know that I can obtain the material I want to sequence. I'm going to start with the silly way, then move onto more sophiticated periplasmic space enrichments, as necessary.
   
3. It could be good to have preliminary co-transformation data to get a more realistic estimate of sequence coverage needed to to our global transformation rate experiments. The biggest help here would be to know the identity of the antibiotic resistance alleles I've been using. And maybe to do whatever it takes to get really high rates...
   

Experimental methods: My first version seemed okay to me, but upon re-reading, it was extremely dry and didn't give too many specifics about the downstream data analysis.

1. Should I keep the experiment using degenerate USS oligos in this grant proposal? It’s a nice experiment, but may require more explanation than I really have space for. I’ve got a better idea now about what the experiment might look like in real life, but it may draw away from the toher components too much to talk about here.
2. I need to more clearly and succinctly discuss the sequencing, particularly distinguishing the difference between “spanning” and “sequence” coverage.
3. The periplasm/cytosol experiments may be real overkill as written. We may want to bring up the possibility of doing competitive experiments between multiple donor genomes, if our single donor experiment goes well. Analysis-wise, it might be nice to show a mock figure of some possible expectations. For example, I discuss but do not illustrate what an uptake blocking sequence would look like.
4. The co-transformation experiment can be improved. Based on the way things are going with the Illumina GA2, we could very well get a lot more out of this than I’d thought. If we could fully sequence 40 independent transformants, we could say a lot...
5. For the bulk transformation experiment, it may make sense to break it into two rounds: In one round, we’d have enough coverage to do a strong analysis of large indel and rearrangement transformation. This could also use a figure or illustration. Since we’ll have high spanning coverage, measuring these rates will use considerably less sequencing cash. If this goes well and we’ve developed a good analytical pipeline, we can then sequence a lot more and pick up the little rearrangements and all the SNPs. At this point, we’d also have a much better idea of how much sequencing we’d need to do to get to whatever level of sensitivity we want to get.

Supplementary essays: Based on the reviews of the grant, the components of my application other than the proposal appear to have been lacking. Here’s some things I need to do to improve these things in the next round:

1. Have a better career plan. I made this essay very short, but the reviewers had some qualms that my research experience did not point at a future coherent research direction. I’ve got plenty of notions in my head about how I might conduct an independent research program, but I need to figure out how to bridge my eukaryotic and prokaryotic research directions together more robustly.
2. Submit 1 or 2 manuscripts from graduate school. I’d said I had two manuscripts in late stages of preparation, which was true. But it’d be nice to actually have these submitted. I’m meeting with my PhD adviser and a co-author next week in California to try and hammer out the last few details of one of the manuscripts. I may still be able to submit the other before the August deadline, since it’s written and only needs some figure-fixing and final edits. But it’s been a long time and will likely take me and my PhD adviser some time to get our brains wrapped around it again.
3. Since I tout my desire to be a teacher, I need to formulate a precise training plan to learn to teach during my postdoc. Rosie has referred me to several possible leads on campus for this purpose...
4. I need to re-orient my research experiences to seem less haphazard. It’s true that after graduate school, I spent a year exploring my options and helping some friends set up their labs, but there was indeed a method to my madness...
5. Letters of support: I need to make sure these letters are perhaps a bit more gushing.

The Style Book. I’m about half way through. It’s mostly filled with things that seem common sense, but reading it has helped me a bit, I think...

(continued...)

Degenerate oligos II

(EDITED to add another graph)

In the first post on our planned degenerate oligo experiment, I described how we might expect the input degenerate oligo pools to look. I used the binomial probability to calculate the chance that a given oligo would have k incorrect bases in an oligo of length n.

So Pn(k) = n! / [ k! * (n-k)! ] * p^(n-k) * (1-p)^k, where p was the probability of the correct base being added at a particular position.

As a thought experiment, I now want to assume that only some of the bases in the n-mer matter to the uptake machinery. I’ll call this number m. The first difficulty was to figure out what the probability of hitting h of the m important bases, given k substitutions in a particular oligo.

This took me a while to figure out. I knew it had something to do with "sampling without replacement" and was able to work out the probabilities by hand for k = 0, 1, or 2, but then I started to get flummoxed. Eventually, I stumbled across the right equation to use: The Hypergeometric Distribution. (That’s seriously cool-sounding.)

Since I don’t know how to write a clean looking equation in HTML (see the above morass given for the binomial probability), I’ll again use the notation ( x y ) to mean "x choose y", or x! / [ y! * (x-y)! ] and will carefully add a * where I mean to indicate multiplication. The hypergeometric is then given as:

f(h; n, m, k) = ( m h ) * ( n-m k-h) / ( n k )

h = hits, or the # substitutions in the important bases
n = total length of the oligo
m = # of important bases in the oligo
k = # of substitutions in the oligo.

So if we define only 1/4 of the bases as important to uptake, then for a 32-mer, m = 8. Then, for a 12% degenerate oligo pool, we get a histogram that looks like this:
If we say that every substitution that hits an important base causes a 10-fold decrease in uptake efficiency, but every hit in am unimportant base causes no change in uptake efficiency, we can then tell what our histogram would look like for both our input oligo pool (IN) and our periplasm-enriched oligo pool (OUT):On the left side of the graph, the output is higher than the input, while on the right side of the graph, the opposite is true. This is what we expect, since the more substitutions in the oligo, the more likely they'll hit important bases.

ADDED:

And here's a third graph addressing Rosie's comment, which shows the ratio of OUT/IN for four sets of conditions:
Here it is again on a log-plot:


(continued...)