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...