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Proof of principle for shotgun DNAmapping by unzipping.
Authors (tentative)
Lawrence J. Herskowitz,lherskow@unm.eduAnthony L. Salvagno,asalvagn@unm.eduR. Andy Maloney,amaloney@unm.eduLinh N. Le,linhle@unm.eduSteven J. Koch,sjkoch@unm.eduAffiliation for all: University of New Mexico, Department of Physics and Astronomyand Center for High Technology Materials.
Abstract
Ability to map polymerases and nucleosomes on chromatin is important forunderstanding the impact of chromatin remodeling on key cellular processes.Current methods (such as ChIP and ChIP-chip) have produced a wealth of information that demonstrates this importance, but key information is elusive inthese ensemble methods. We’re pursuing a new single-molecule chromatinmapping method based on unzipping native chromatin molecules with opticaltweezers. The first step we are taking towards this ability is shotgun DNA mapping(SDM). This is the ability to identify the genomic location of a random DNAfragment based on its naked DNA unzipping forces compared with simulatedunzipping forces of a published genome. We show that ~32 separate experimentalunzipping curves for pBR322 were correctly matched to their simulated unzippingcurves hidden in a background of the ~2700 sequences neighboring XhoI sites inthe S. cerevisiae (yeast) genome. We describe this method and characterize itsrobustness as well as discuss future applications.
Abbreviations
SDM = Shotgun DNA Mapping; SM = single-molecule; ChIP = ChromatinImmunoprecipitation; Pol II = RNA Polymerase II; SCM = shotgun chromatinmapping
Introduction
(
1-6
)
Chromatin remodeling affects the ability of other proteins to access the DNA andhas a profound impact on fundamental processes such as DNA repair and genetranscription by RNA polymerase. Understanding of these dynamic remodelingprocesses requires the ability to characterize with high spatial and temporal
 
resolution the changes to chromatin inside living cells. Techniques such aschromatin Immunoprecipitation (ChIP), ChIP-chip, and other existing techniqueshave provided a wealth of important information, but have drawbacks in terms of sensitivity to small changes in protein occupancy, spatial resolution, and ensembleaveraging. Certain information can only be obtained via single-molecule (SM)analysis, such as seeing direct correlations between polymerases and nucleosomeson individual fibers or differentiating between some proposed models of chromatinremodeling
(
7
)
. To obtain this type of information, we are developing a single-molecule method formapping polymerases and nucleosomes on chromatin based on optical tweezersunzipping of native chromatin molecules. It has been shown that SM DNAunzipping can map the positions of mononucleosomes assembled in vitro based ona repeatable nucleosome unzipping force profile
(
8
)
. We expect RNA Polymerase II(Pol II) complexes to also have a repeatable unzipping force profile, butdistinguishable from nucleosomes and perhaps also indicating the sense / antisenseorientation of the Pol II. The Pol II data is not yet available, but if it is as expected,then we anticipate that SM unzipping of native chromatin fragments (extractedfrom living cells) will provide high-resolution mapping of nucleosomes and Pol IImolecules (along with orientation) on individual chromatin fibers.
Figure 1Overview of proposed method for shotgun DNA and chromatin mapping. We have recentlyachieved proof-of-principle results important for the “Global Genome Location,” part of theprocess (
lower right 
).
We may be able to obtain important information from high-resolution SM mappingon individual fragments, even if their specific location in the genome were unknown.For example, the electron microscopy analysis of chromatin and RNA transcripts hasdemonstrated the utility of SM information even when the identity of the genes wasunkown
(
9
)
. However, it would be much more powerful and thus desirable to obtainhigh-resolution SM information about specific genes or other sites in the genome of interest. For example, site-specific SM analysis may provide crucial insight into the
 
issues of promoter-proximal Pol II pausing and antisense transcription which haverecently been shown to be very important
(
10-16
)
. Thus, we are pursuing methods for
site-specific
SM analysis of chromatin. The first way we have tried to do this is byengineering unique restriction sites into the yeast genome (I-SceI) at a specific site. This has proven difficult, and has the disadvantage of requiring genetic engineeringof all mutant strains and cell types that will be analyzed. Thus, we are nowpursuing a second way of achieving site-specificity which is to unzip randomchromatin fragments in a high-throughput fashion, and then figuring out from whichspecific site of the genome it came. We call this shotgun chromatin mapping (SCM)and it based on a method for indentifying the genomic location of naked DNAfragments (see Fig. 1).It has been shown that the unzipping forces for a known sequence of DNA can beaccurately predicted by statistical mechanical models
(
17, 18
)
. Furthermore, at thistime many genomes have been published and the number is rapidly increasing. These two facts together led us to believe that the naked DNA experimentalunzipping forces would allow us to identify the genomic location of random DNAfragments. We call this process shotgun DNA mapping (SDM). The basic procedureis to compare an unknown fragment's force data to a library of known possiblefragments’ simulated unzipped force data. The fragment possibilities can belimited, for example, by digestion with a site-specific restriction endonuclease. In asuccessful method, the experimental data will reliably match up the best with thesimulation of its true sequence. The identify of a DNA fragment could be easilyidentified manually (“by eye”) from among a handful of possibilities, but it remainsto be shown whether the simulations can be accurate enough for automatedidentification a fragment from the background of thousands of fragments expectedfrom site-specific digestion of genomic DNA.In this paper, we show that SDM is possible. Specifically, we demonstrate that themodeling of the pBR322 unzipping forces is sufficiently accurate so thatexperimental data are successfully matched to the pBR322 sequence hidden in abackground of the ~2700 XhoI fragments from the yeast genome. We explain ourmethods, show where to obtain our software and data, and discuss further potentialimprovements which indicate it will also be successful with much larger fragmentlibraries. We feel this technique will be a key enabler of our goal of shotgunchromatin mapping. Furthermore, we envision other high impact applications, forexample single-molecule structural genome mapping
(
6
)
and new assays forscreening protein binding sites by shotgun DNA mapping in the presence of purifiedproteins.
Methods
All computations below were carried out using a [Dell duoCore running Windows XP.Code written in LabVIEW 7.1
of 00

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reply12 / 25 / 2010

uploaded a new revision for this document (#2)

12 / 15 / 2009
sjkoch4914

Thanks Richard! We've responded to many of your questions / comments on the OpenWetWare page: http://openwetware.org/wiki/Koch_Lab:...

reply01 / 25 / 2009
sjkoch4914

Thanks, Richard! Your suggestions are really helpful and inspiring. We'll get back to you within a few days.

reply01 / 21 / 2009
sjkoch4914

We forgot to acknowledge Ben Jantzen. Larry developed his simulation algorithms from a blank page, but we did look at the code Ben created way back in 2001 or whatever, and that was helpful.

reply01 / 21 / 2009
richard.c.yeh372

First, congratulations on your new paper draft! My understanding of your paper is that you have shown that you can identify a naked dsDNA sequence by comparing its unzipping force-index curve against a library of simulated curves. Very cool, and I want to thank your team for writing the paper in a way that I can understand. Here are my comments: 1. I think the major contribution of the paper is that you have an accurate simulation and matching method, which can easily account for elasticity of the tethering construct, viscosity, etc. I'd like to see more details (or at least know that you have the answers in your back pocket) on the information-recall side of the problem. See terms at the bottom of < http://en.wikipedia.org/wiki/Sensitiv... >. I'm no expert here, but here are my thoughts (and most of the answers don't necessarily belong in the paper): You matched experimental F-j curve vs simulated F-j curve. Why not interpret your experimental F-j curve into a sequence (with wild-card characters) and then do the matching in ASCII space? Is there a rough map between your match score and a BLAST similarity score? (But, since I'm no BLAST expert, I would need to do lots more research before talking about BLAST in the paper.) In the "Future Improvements" section, you discuss the general sequencing-by-unzipping problem. Imagine simulating all possible sequences from j=1200 to j=1700. In this space of 4^500 sequences, what are the characteristics of those close to your experimental sequence, and what is the minimum similarity that can be resolved? From the other side, in your matching problem, how much easier has the problem been made by considering only the 2700 restriction fragments instead of the 4^500 general genome? What if you don't restrict yourself to those restriction fragments, but allow any contiguous 500-bp sequence from yeast --- then how many false positives do you get? (In Figure 4, what is the outline that you would get by simulating random fragments from the 4^500 or yeast-sequence universe?) A related question is characterizing the sequences in the overlap of the Gaussians. Suppose when you unzip chromatin, you get only a naked-DNA signal over the linker DNA regions, and an un-simulated signal over the non-linker regions. Would you speculate on how that (change in signal) will affect your recall? Suppose you simulate all 2700 restriction fragments. What if you make another version of figure 4 by scoring all simulated sequences versus each other (sim vs sim instead of expt vs sim)? (Also, by symmetry, what is the spectrum of scores obtained for individual OT runs --- both match and mismatch --- against each other (expt vs expt, instead of expt vs sim)?) Is the difference in distance between your blue and red peaks roughly equal to the difference in distance between correct and incorrect peaks for the 2700 x 2700 test? (If not, then you will have to explain what's special about your 32 sequences.) Do the 2700 fragments include the reverse-complement sequences? 2. In the excerpt of the match score formula, I see kT / C / stdev(force difference). Here are the implications that I read from the formula: a. for a given match score, a 1-unit force difference on 4 indices (neighboring or separate) is worth a 2-unit force difference on 1 index; and b. for a given match score, the standard deviation of the force difference scales with the temperature of the environment. On (a), I recognize that this is a draft and see that you may not want or need to refine the formula. On (b), are you making a statistical-mechanical statement (and I see no support for it elsewhere in the paper), or are you just dressing up the standard deviation of force in statistical-mechanical clothing, by adding a kT and making it unitless? For the purposes of your experiment, kT/C is constant, so that portion of the relationship cannot be tested. My thought is, if you're not making a statistical-mechanical statement, then your match score should be as simple as possible: m2 = stdev(force difference/scale force) where the scale force is kT/2/C. Do you actually get a better Gaussian when you take exp(-1/m2)? My own biases would reverse your scale, suggesting numbers close to 1 as a good match, and numbers close to zero as a poor match --- maybe something that can be interpreted as a probability, like exp(-m2) / sum_{all sequences} exp(-m2). 3. The beginning of the paper reads to me like "peeing on the tree". In particular, I think mentioning Pol II is overreaching. 4. It really bothers me in figure 2 that the colors are swapped between A and B. Also, next to figure 4, in the last paragraph before "Future Improvements", you use the word "fell" instead of "felt". Also, I have not worked on the language very much, but the last line on page 8 is a little strange. 5. You mention viscosity but don't explicitly say whether your modeled force includes viscosity. I am going to guess that it does, because otherwi

reply01 / 20 / 2009
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