Return from ASM

Just got back from the ASM meeting in Orlando, FL. The meeting was good - amazing things being done with full genome comparisons, plus microarray gene expression analyses. Anyway, just a few notes here:

1) A new software package called CLC that is definately worth investigating. [http://www.clcbio.com/]

2) A product called Perfect Match® PCR Enhancer from Statagene. Here is the manual.

See an article by De Milito et al. 1995. I also found this comment in a molecular biology group:

"[Perfect match is] Tetramethyl Ammonium Chloride. It costs about $30 for 500 gms from Fisher, and stinks to high heaven.I titrated it between 10 uM (micromolar) and 1 nanomolar leaving allother conditions the same."

This is not verified information.

Cheers,
Stefan

Update

Well, good news. Another manuscript of mine has been accepted for publication in the journal Applied and Environmental Microbiology. Wooh! I just sent the galley proofs back to the editor. It really looks great. I had a very positive experience with AEM - not just because my manuscript was accepted. The peer-review was excellent - great commentary by two reviewers and by the editor as well. After acceptance, the page proofs were generated very rapidly, and I'm happy to say that the manuscript has been published. Reference:

Stefan J. Green, Ehud Inbar, Frederick C. Michel, Jr., Yitzhak Hadar, and Dror Minz. 2006. Succession of Bacterial Communities during Early Plant Development: Transition from Seed to Root and Effect of Compost Amendment. Appl. Environ. Microbiol. 72:3975-3983.


Anyway, things are moving along here at NASA. We finally received all the various bits of our real-time PCR machine from Bio-Rad. Since I still have no reagents, I haven't really been able to check out if the thing is working or not. But it looks nice! Also, a third sulfate experiment has been begun and I've gathered some microbial mat samples at the initial time point (before removal of sulfate). I'm hoping to examine microbial communities at different depths during a diurnal cycle in the mat.

The summer is shaping up to be very busy. Meeting in Orlando next month (ASM) and Vienna in August (ISME). Plus, I am co-advising a student working on microbial communities associated with ophiolites. Should be interesting research. However, my position is very much in flux as my fellowship is expiring at the end of September this year. More on the state of affairs as it becomes clear.

A publication of interest: A computer program ("TreeClimber") to analyze microbial communities via analysis of phylogenetic trees. This seems similar to UniFrac, but I haven't examined it closely. However, the documentation seems better than UniFrac. Find the article here. Find the website here.

Finally, I note that NCBI has started a "Probe" database. Should be very useful.

Cheers,
Stefan

UniFrac Analysis

Well, just a note of some interesting articles regarding new statistical techniques to analyze sequence libraries in a manner akin to analyzing DGGE profiles:

Lozupone, C. and R. Knight. 2005. UniFrac: a New Phylogenetic Method for Comparing Microbial Communities. AEM: 71: 8228-35.

And an application of UniFrac:
Ley, R.E. et al. 2005. Obesity alters gut microbial ecology. PNAS. 102:11070-11075.

To use UniFrac, you will need to download and operate "Python" software.
You will also need to download a special "Numeric" module. Make sure you download the Numeric, not the NumPy, module.
Then you will need to download the .zip file of the unifrac program (http://bayes.colorado.edu/unifrac.zip).

The UniFrac program is not trivial to use if you haven't had any experience with Python or similar languages. I've emailed Dr. Lozupone and she has indicated that eventually a web-based UniFrac program will be available. However, not yet.

You will need phylogenetic trees in Newick tree format as the input data file for this program.

DGGE Help Page

I am announcing the release of my DGGE Help Guide. I have spent some time reviewing the questions that have accumulated on the YahooGroups DGGE page and decided to compile a guide to answer many of the questions that come up repeatedly. The guide is not meant to be definitive, but it is meant to be a resource for users to find the information that they do need. I also put in some of my own personal opinions regarding PCR and DGGE.

I hope that people find the guide useful. I will update the guide as time goes along, and I will be happy to receive additional information from anyone interested in providing it. If anyone feels that there are sections that they would like to write, update or correct, please contact me.

I have the guide on another blog: http://ddgehelp.blogspot.com/
The entire text can be had in word format or PDF format. However, I have found that the PDF format does not retain the hyperlinks. For the intended format, please use the word file.

Monthly Update

Well, it seems about time for a monthly update. I had thought I might do this more routinely, but I guess this is what we can expect! I have added a few tidbits to the SuPER PCR page. These include citations to two very nice manuscripts. One is a method similar to SuPER PCR but for RNA and the other is a clever mechanism to circumvent the problem of plastid DNA in environmental studies of plant roots.

Things here are going well. I am in the midst of a major sequencing effort relating to the "Sulfate II" experiment that was conducted at NASA. The idea was to take hypersaline microbial mats (See Bebout et al. 2005; Minz et al. 1999 and many others) and incubate them under controlled conditions at standard salinity (85 ppt), lowered salinity (35 ppt), at standard sulfate concentrations (~60 mM) and at low sulfate concentrations (< 1 mM). After letting these mats acclimate to these conditions, a suite of geochemical measurements were taken (oxygen/sulfide profiles, methane evolution, etc), and I have been working, together with Jason Smith (U of Florida), Mary Hogan (NASA/ARC), Brad Bebout (NASA/ARC) and Lee Bebout (NASA/ARC), on analyzing the methanogens and sulfate reducers in the system. The preliminary analyses of the methanogens are quite striking and indicate a dramatic shift in population as a result of the removal of sulfate. More on that when all the data are in. No sequence data yet on the sulfate reducers.

I have also been performing some phylogenetic analyses with the program Mr.Bayes (discussed in a prior posting). However, I am having problems with the technique - with regard to the branch lengths that the method is yielding. I have noticed that the branchlengths produced by MrBayes are significantly longer than those I get from other treeing methods such as Neighbor Joining or Maximum Likelihood. I have observed that the branch lengths are roughly 2x those of NJ, and 2x thes equence difference (as estimated by BLAST or other aligning method). I realize that MrBayes branch lengths are "expected changes per site" and not necessarily the same as NJ branch lengths. Still, the 2x factor seems excessive. If anybody has any thoughts on this I would love to hear them. Overall, the branching order yielded by Mr. Bayes seems quite consistent with other methods.

Well, more soon.

Contract Renewal

Well, good news here. My NRC contract was renewed for a second year. It's nice to be employed. However, since the NRC fellowship is essentially limited to two years, it is time to start looking for another position. I have applied for some grants as well, but it will be quite a while before I hear about the results of those applications.

Things are starting to get quite busy. I have a number of sequencing projects to support a large amount of geochemical data acquired in a long-term project here at Ames. The projects involved adding nutrients (ammonia,nitrate or phosphorus) to microbial mats maintained in the greenhouses here, and a second major experiment in which salinity and sulfate levels were modulated. So, we are examining major microbial functional groups: methanogens, sulfate-reducers, photosynthetic populations, methane oxidizers, and nitrogen-fixers. More updates on the results of these experiments soon.

Also, happy new years (shana tova) and happy birthday (to me).

GenomiPHI Update

I've done a little more work with the GenomiPHI kit. It is not a panacea for all that ails with PCR done directly on environmental DNA. I have had mixed results with different samples. Some samples with weak PCR do not fare well with the kit while others do. I have also determined that halving the recommended volumes does not help - you should use their recommendations.

On the plus side, I performed a denaturing gradient gel electrophoresis (DGGE) analysis of an environmental DNA extract directly and after pre-amplification with the genomi-PHI kit. The bacterial 16S ribosomal RNA gene profiles look identical before and after pre-amplification, suggesting that the pre-amplification is not biased.