Psyched: have rescued old MobileMe and other websites after Apple annoyingly cancelled them by posting to Dropbox

A few years ago I used to post many things for the Web through Apple’s Mobile Me service.  Annoyingly, Apple ended up treating this like they treat connectors and plugs for their phones and Macs.  They just decided to move their online system to iCloud and deleted all the old websites through Mobile Me.  Which left me in a lurch.  And then I forgot about it.  But I have been rediscovering how annoying this is since I had a lot of information out there on old papers and projects and now it is gone from the interwebs.  So I have ben trying to re-share all of this stuff.

One way has ben to post data from old papers to Figshare.  See for example:

But I also had all sorts of website related material that is annoyingly gone.  And yesterday I discovered at least a simple solution to this.  I can put all my old websites in my Dropbox public folder and share the link to those files with others and they work pretty well.

See for example my re-releasing of some of my April 1 and other joke websites:

 Also – I have reposted some of the my old websites

I have always been into sharing scientific information on the web since, well, the web came out.  And I am going to dig around for other old websites to post them via Dropbox.  If anyone knows an easy way to upload / convert an old website into WordPress, I suppose I could load in all the old pages into my current wordpress site, but this was a much easier temporary solution.  Still annoyed with Apple but glad Dropbox allows a simple solution.

The Axis of Evol: Getting to the Root of DNA Repair with Philogeny

The Axis of Evol: Getting to the Root of DNA Repair with Philogeny 
In 2005 I wrote an essay about my time in graduate school that was potentially going to be included in a special issue of Mutation Research in honor of my PhD advisor Phil Hanawalt.  Alas, publishing my essay ran into complications in regard to the closed access policies of this journal.  So in the end, my essay was not published.  I had forgotten about it mostly until very recently.  And so I decided to convert the essay to a blog post.  The essay is sort of about what I did in grad. school and sort of about Phil …
Phylogenomics is a field in which genome analysis and evolutionary reconstructions are integrated. This integration is important because genome data is of great value in evolutionary reconstructions, because evolutionary analysis is critical for understanding and interpreting genomic data, and because there are feedback loops between evolutionary and genome analysis such that they need to be done in an integrated manner. In this paper I describe how I developed my particular phylogenomic approach under the guidance of my Ph.D advisor Philip C. Hanawalt. Since I was the first to use the term phylogenomics in a publication, I have decided to rename the field (at least temporarily) Philogenomics.
1. Doctor of Philosophy
When I went to Stanford for graduate school, I was interested in combining evolutionary analysis and molecular biology in a way that would allow me to study molecular mechanisms through an evolutionary perspective. Although I had gone to Stanford ostensibly to work on butterfly population genetics, within two days of starting a rotation in Phil’s lab, I knew that that was where I wanted to work. This decision was somewhat traumatic, since the work on butterflies included spending the summers at 10,000 feet in the Rocky Mountains and possibly chasing butterflies like a Nabakov wanna-be all over the mountain ranges of the world. As an avid outdoor person, this was quite appealing. Nevertheless, I chose to spend 99% of my graduate work in the dingy confines of Herrin Hall, studying DNA repair. The choice of joining Phil’s lab did have one very positive affect – and that was on my relationship with my grandfather on my mother’s side. Benjamin Post was in many ways like a father to me, especially after my father passed away. He was a physicist from the “old school” and thought that most of biology was completely useless. Needless to say, when I told him I was going to graduate school in California (which he considered already one strike against me) to study butterflies, he decided I was simply a lost cause. Despite all his talk of Einstein and computers and math when I was a child, I might as well have been a poet from his point of view. To make matters worse, my grandfather was a crystallographer, and my brother was getting his Ph.D in crystallography at Harvard. When I informed my grandfather that I was going to be working on DNA repair, he seemed somewhat interested. And then I told him, my advisor, Phil Hanawalt, is relatively well known, and actually used to be considered a biophysicist. Then my grandfather really perked up. He said, “Hanawalt – is he related to Don Hanawalt?” It turns out, that my grandfather worked in the same field as Phil’s father (they both did powder diffraction) and knew him. So my grandfather said “You may not be doing real science, but at least you are doing it with the relative of a real scientist.” Thankfully, I was no longer the black sheep in the family. So, with my grandfather’s approval, I embarked on a career in DNA repair.

I would like to add that I was very torn in writing this article. On the one hand, Phil was the greatest advisor I could ever imagine, allowing me to pursue studies on the evolution of DNA repair and comparative genomic analysis, even though nobody else in the lab worked on such things and at times, nobody seemed interested in them either. Phil’s support allowed me to explore my own interests and develop my concepts for the idea of “Phylogenomics” or the combining of evolutionary reconstructions and genome analysis. On the other hand, this special issue is being published in an Elsevier journal. As a supporter of the Open Access movement on scientific publications (see and the brother of one of the founders of the Public Library of Science, publishing in an Elsevier journal is like cavorting with the devil. But the pull of Phil is very strong (some strange sort of force actually) and despite the effects that this may have on my relationship with my brother, I have agreed to publish in this special issue, and thus can now say that I sold my soul for Phil Hanawalt. [[OOPS – Spoke too soon on this when I wrote it — in the end I just could not sign on the dotted line]].
In this essay, I describe my development in Phil’s lab of the idea of “Phylogenomics” or the combination of evolutionary reconstructions and genome analysis. I would like to add that this is not an attempt to review the field of phylogenomics or all the studies that could be called phylogenomics of DNA repair. For that I recommend reading other papers by myself (some of which are discussed below) as well as those by Rick Wood [1-4]}, Janusz M Bujnicki [5], Eugene Koonin [6-14]}, Carlos Menck [15-18], Michael Lynch [19-21], Patrick Forterre [22-24], Nancy Moran [25-29], and others. This is just meant to review my angle on the phylogenomics of repair and Phil’s contribution to this.
2. RecAgnizing the value of evolutionary analysis in studies of DNA repair
A post-doc in Phil’s lab at the time I was there, Shi-Kau (now known as Scott) Liu was working on analysis of some studies of recA mutants he had done while working in Irwin Tessman’s lab. He asked me if I could help him with some comparative analyses of RecA protein sequences from different species, in the hopes that this might help interpret his experimental data. We then downloaded and aligned all available RecA protein sequences from different species of bacteria and compared the sequence variation to the recently solved crystal structure of a form of the E. coli RecA protein. Specifically we were looking for compensatory mutations in which there was a change in one amino-acid in the region there was a correlated change in another amino-acid in the same region (these were detected using an evolutionary method called character-state reconstruction).  Interestingly, in some regions of the crystal structure (e.g., the monomer-monomer contact regions) extensive compensatory mutations could be detected, suggesting that this region of the crystal was conserved between species. In other regions of the crystal (e.g., the filament-filament contact regions), no compensatory mutations could be detected suggesting either that this region of the structure was not conserved between species or that the filament contact regions were some artifact of crystallization. This was important to show since the mutations Shi-Kau was looking at were suppressors of another recA mutant (recA1202) and the suppressors we found did not make complete sense if the filament-filament contact regions of the crystal reflected perfectly what was going on in-vivo (30).
In this way, evolutionary reconstructions helped inform experimental studies in E. coli. While this concept was not necessarily novel, it is important to point out that most molecular sequence comparisons used for structure-function studies both then and now focus on sequence conservation (that is, what is identical or similar between sequences). This does not take full advantage of the evolutionary history of sequences since it does not specifically examine how the sequence conservation came to be (that is, it does not look at the amino-acid changes that occurred, just what is conserved). This made me realize that comparative analysis (identifying what is similar or different between genes or species) was fundamentally different from evolutionary reconstructions (which can identify how and possibly even why the similarities and differences came into being). I should point out that to do the compensatory mutation analysis well requires lots of sequences and this was one of the hidden reasons behind why I have pushed for ten years for people studying the evolutionary relationships among microbes to use recA as a marker as they use rRNA (31).
3. Sniffing around at homologs of repair genes
Shortly after the recA analysis was complete, another problem being addressed in the Hanawalt lab presented an even more powerful test for evolutionary reconstructions. Kevin Sweder, another post-doc in the lab, was working on yeast strains with defects in homologs of human DNA repair genes. It was at this time that many of the human DNA repair genes were being cloned and shown to be members of the helicase superfamily of proteins. Many of these could further be assigned to one particular subfamily within the helicase superfamily – the subfamily that contained the yeast SNF2 protein. Proteins in the SNF2 family could be readily identified because their helicase-like domains were all much more similar to each other than any were to other helicase-domain containing proteins. Yet many scientists, including Kevin, were presented with a problem. As the yeast genome was being completed, blast searches could identify that yeast encoded many proteins in the SNF2 family. However, these same blast searches could not readily identify which yeast gene was the orthologs of which human gene. For those who do not know, homologous genes or proteins come in two primary forms – paralogs, which are genes related by gene duplications (e.g., alpha and beta globin) and orthologs, which are the same form of a gene in different species (e.g., human and mouse alpha-globin). Thus if one wanted to use yeast as a model to study a human disease due to a mutation in a SNF2 homolog, it would be helpful to know which yeast gene was the ortholog of the human gene of interest. Since paralogs are related to each other by duplication events and since duplication events are an evolutionary event, I figured that an evolutionary tree of the SNF2 family proteins might help divide the gene family into groups of orthologs.
Indeed, this is exactly what we found – the SNF2 family could be divided into many subfamilies, each of which contained a human and a yeast gene and thus these genes could be considered orthologs of each others. In our analysis we found something even more striking. For every subfamily in the SNF2 superfamily, if the function of more than one member of the subfamily was known (e.g., the human and yeast genes), the function was always conserved. Also, all different subfamilies appeared to have different functions (32). Thus one could predict the function of a gene by which subfamily in which it resided. As with the analysis of RecA, it should be pointed out that the phylogenetic tree-based assignment of genes to subfamilies was more useful than blast searches because blast is simply a way to identify similarity among genes/proteins. The tree allows one to group genes into correct subfamilies even if rates and patterns of evolution have changed over time and are different in different groups. Again, this is a distinction between comparative analysis and evolutionary analysis.
4. A gut feeling leads to the idea of “Phylogenomics”
With the SNF2 analysis as a backdrop, I proceeded to proselytize to anyone who would listes, that phylogenetic trees of genes were going to revolutionize genomic sequencing proteins by allowing one to predict the functions of many unknown genes. Genome sequencing projects of course product lots of sequence data and little functional information. Although most of the people in the Hanawalt lab (except maybe Phil) could not have cared less about my evolutionary rantings, fortunately for me, one person called my bluff. Rick Myers, a professor in the Stanford Medical School and one of the heads of the Stanford Human Genome Center was asked to write a News and Views for Nature Medicine about the recent publications of the genomes of E. coli O157:H7 and Helicobacter pylori. So Rick challenged me and said I should try and come up with a real example of how the people who worked on these genomes screwed something up by not doing an evolutionary analysis. Fortunately, it was easy to find an interesting case to study in one of the genomes. In the H. pylori paper, the authors had predicted that the species should have mismatch repair but then reported something quite unusual – the genome encoded a homolog of MutS but did not encode a homolog of MutL. I suppose this should have raised a red-flag to them since all species known to have mismatch repair required homologs of both of these proteins for the process. While some species had other bells and whistles (e.g., the use of MutH and Dam in gamma proteobacteria), the use of MutS and MutL was absolutely conserved. An evolutionary tree of the MutS homologs available at the time including the one in H. pylori also suggested a red-flag should have been raised before predicting that this species possessed mismatch repair.
The MutS family in prokaryotes could be divided into two separate subfamilies, which I called MutS1 and MutS2. All genes known to be involved in mismatch repair were in the MutS1 family. No gene in the MutS2 family had a known function. The H. pylori gene was in the MutS2 family. So this species had no MutL and a MutS homolog in a novel subfamily. To us, this suggested that it would be a bad idea to predict the presence of mismatch repair in this species (33). Later, I showed that there was a general trend – all prokaryotes with just a MutS2-like protein did not have a MutL-homolog, and all species with a MutS1-like protein did (34-36). Experimental work has now shown that the MutS2 of H. pylori is not involved in MMR and that this species apparently does not have any MMR (37). This is important because this apparently causes this species to have an exceptionally high mutation rate, which in turn can effect how one designs vaccines and drugs and diagnostics to target it. It should be pointed out that the role of the MutS2 homologs is not known although they have been knocked out in many species and as of yet none have a role in MMR. Thus predicting function by evolutionary analysis (or more specifically, not incorrectly predicting function) can be of great practical value.
      It is from this analysis that I came up with the idea of “Phylogenomics” or the integration of evolutionary reconstructions and genome analysis (34-36). These approaches should be fully integrated because there is a feedback loop between them such that they cannot be done separately. For example, in the studies of MutS and MutL it is necessary to do a genome analysis to identify the presence or absence of homologs of these genes, then an evolutionary analysis to determine which forms of each of the genes are present, then a genome analysis again to determine the number and combination of different forms and then an evolutionary analysis to determine whether and when particular forms were gained and lost over evolutionary time, and so on. 
5. Lions and TIGRs and bears
Since leaving Phil’s lab I have been a faculty member at The Institute for Genomic Research (TIGR) and in that time we have found dozens of new uses for a phylogenomic approach and designed many new methods to implement phylogenomics. Such an approach has led to many interesting findings relating to DNA repair. Phylogenetic analysis of eukaryotic genomes has allowed us to identify many nuclear encoded genes that are homologs of DNA repair genes but appear to evolutionary derived from the organellar genomes and thus are good candidates for still having a role in DNA repair in the organelles (38). These include both putatively plastid-derived genes (encoding RecA, Mfd, Fpg, RecG, MutS2, Phr, Lon) and mitochondrial-derived genes (encoding RecA, Tag). Interestingly the presence of Mfd but not UvrABCD is also found in many endosymbiotic bacteria, although the explanation for what this Mfd might be doing is unclear. Phylogenomic analysis has allowed us to identify the loss of important DNA repair genes in various species such as the apparent loss of all the genes for non-homologous end joining in the causative agent of malaria, Plasmodium falciparum (39). An important component of this analysis was the finding that this species did not have an orthologs of DNA ligase IV, even though the original annotation of the genome had suggested it did (Figure 1). 
Figure 1. Phylogenetic tree of DNA ligase homologs showing the presence of an orthologs of DNA Ligase I in Plasmodium falciparum but no orthologs of DNA ligase IV, consistent with the absence of non homologous end joining. 
Among the other interesting repair-related features we have found are: the presence of two MutL homologs in an intracellular bacteria Wolbachia pipientis wMel (40), the presence of two UvrA homologs in Deinococcus radiodurans (41) and Chlorobium tepidum (42), the absence of MutS and MutL from Mycobacterium tuberculosis(43), and the presence of multiple ligases for each chromosome in Agrobacteriumtumefaciens (44). Continued surprises come from almost every genome.
However, all is not good in the world of phylogenomics. One of the biggest problems is that most of the experimental studies of DNA repair that have formed the basis of out knowledge in the field have been done in a narrow range of species. For example, there are estimated to be over 100 major divisions of bacteria (Phyla) and of these, most DNA repair studies have been restricted to three of these phyla (Proteobacteria, Firmicutes (also known as lowGC Gram-positives), and Actinobacteria (also known as highGC Gram positives). This means that if anything novel evolved in any of the other lineages, we would not know about it. This probably explains why, when we sequenced the genome of the radiation resistant bacteria D. radiodurans, analysis of the homologs of DNA repair genes in the genome did reveal many homologs of known repair genes but this list did not have many features that were unusual compared to non radiation resistant species (Table 1) and thus was not of much use in understanding what makes this species so resistant (41).
Table 1. Homologs of known DNA repair genes identified in the initial analysis of the D. radiodurans genome sequence
Genes in D. radiodurans
Unusual features
Nucleotide Excision Repair
UvrABCD, UvrA2
UvrA2 not found in most species
Base Excision Repair
AlkA, Ung, Ung2, GT, MutM, MutY-Nths, MPG
More MutY-Nths than most species
AP Endonuclease
Mismatch Excision Repair
MutS, MutL
   Migration and resolution
RuvABC, RecG
PolA, PolC, PolX, phage Pol
PolX not in many bacteria
dNTP pools, cleanup
MutTs, RRase
LexA, RadA, HepA, UVDE, MutS2
UvDE not in many bacteria

 This of course means that genome sequencing and analysis, even if done in a robust way, only works well if there is a core of experimental studies on which to base the analysis.
In the end, I would like to define a new word – philogenomics which is the combination of studies of evolution, genomics, DNA repair, thymine metabolism, and punning. The ultimate proof of a philogenomic approach, of course, will come when it figures out the mechanism underlying thymineless death. But that is another story.
6. Acknowledgements
I would like to thank Philip C. Hanawalt for his support during and after my Ph.D research in his lab. Everyone in the field knows he is a great scientist. What they may not all know is that he is an even better human being.
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Draft post cleanup #12: RecA is cool (and interesting)

Yet another post in my “draft blog post cleanup” series.  Here is #12:

I have been interested in RecA and related proteins for many many years.  In particular I have been interested in structural and functional evolution of RecA and its relatives.  This all started when for my second scientific paper I helped a post doc in the lab where I was doing my PhD do some structure-function-evolution studies (with a little help from Chris Lee, then in Mike Levitt’s lab, and my brother, then in Don Wiley’s lab).

For my first talk at a scientific meeting I discussed using RecA as a marker for phylogenetic studies (and had a slide where I had text saying RecA was cool).  Over the years I have continued to try and study RecA or at least use it for studying microbial diversity in some way.  See for example

Anyway – in this context I was excited to see a new paper on RecA-structure-function-evolution studies: PLoS Genetics: Separation of Recombination and SOS Response in Escherichia coli RecA Suggests LexA Interaction Sites from Olivier Lichtarge and others.  In the paper they use Lichtarge’s Evolutionary Trace method to study RecA.

The paper is worth a look and if you are interested in structure-function-evolution types of studies and need a good protein to work on, I would suggest you look at RecA and its relatives … They are Cool.

Oh and for the fun of it — I have found some of my slides from that talk in 1995.  Here they are

The story behind the story of my new #PLoSOne paper on "Stalking the fourth domain of life" #metagenomics #fb

Well, here goes.

This is a post about a paper that has been a long long time coming. Today, a paper of mine is being published in PLoS One. The paper is titled “Stalking the Fourth Domain in Metagenomic Data: Searching for, Discovering, and Interpreting Novel, Deep Branches in Marker Gene Phylogenetic Trees” and is available at (or if that link does not work you can get a copy here). This paper represents something I started a long time ago and I am going to try to describe the story behind the paper here.

I note – we are not doing a press release for the paper, for a few reasons. But one of them is that, well, I am starting to hate press releases. So I guess this is kind of my press release. But this will be a bit longer than most press releases. I note – my key fear here is that somehow in my communications with the press or in our text in the paper or in this post I will overstate our findings. Here is the punchline – we found some very phylogenetically novel forms of phylogenetic marker genes in metagenomic data. We do not have a conclusive explanation for the origin of these sequences. They may be from novel viruses. The They may be ancient paralogs of the marker genes. Or they may be from a new branch of cellular organisms in the tree of life, distinct from bacteria, archaea or eukaryotes. I think most likely they are from novel viruses. But we just don’t know.

UPDATE: Am posting some links here to news stories/blogs about our paper

    First – a summary of what we did.

    In the paper, we searched through metagenomic data (sequences from environmental samples) for phylogenetically novel sequences for three standard phylogenetic marker genes (ss-rRNA, recA, rpoB). We focused on sequences from the Venter Global Ocean Sampling data set because, well, we started this analysis many years ago when that was the best data set available (more on this below). What we were looking for were evolutionary lineages of these genes that were separate from the branches that corresponded to the three known “Domains” of life (bacteria, archaea and eukaryotes).

    To search for such novel lineages in the metagenomic data, we built evolutionary trees using these genes where we included sequences from known organisms (and viruses) as well as sequences from metagenomic data. We then looked through the trees for groups that were both phylogenetically novel and included only environmental data (i.e., they were new compared to known organisms or viruses). This method did not work very well for rRNA sequences (largely because making high quality alignments of short phylogenetically novel rRNA sequences was difficult – more on this below). But with RecA and RpoB homologs we were able to generate what we believe to be robust phylogenetic trees. And in these trees we found evidence for phylogenetically very novel sequences in environmental data.

    Figure 1. Phylogenetic tree of the RecA superfamily. 

    Figure 3. Phylogenetic tree of the RpoB superfamily

    We then propose and discuss four potential mechanisms that could lead to the existence of such evolutionarily novel sequences. The two we consider most likely are the following

    1. The sequences could be from novel viruses
    2. The sequences could be from a fourth major branch on the tree of life

    Unfortunately, we do not actually know what is the source of these sequences. So we cannot determine which of the theories is correct. Obviously if there is a novel lineages of cellular organisms out there, well, that would be cool. But we have no evidence right now if that is what is going on. Personally, I think it is most likely that these novel sequences are from weird viruses. But as far as we can tell, they truly could be from a fourth major branch of cellular organisms and thus even though we did not have the story completely pinned down, we decided to finally write up the paper to get other people to think about this issue.

    Below I give all sorts of other details about the project in the following areas

    • The history of the project 
    • More detail on what is in the paper 
    • Follow up analysis and rapid posting with google Know 
    • Data deposition in Dryad 
    • Who was involved 
    • UPDATE: Funding for this work

    The history of the project

    Well, this is one of those projects for which the history is hard to explain. We started this work in 2004 when I was helping Venter and colleagues analyze the Sargasso Sea metagenome data. I was working at TIGR in 2003, which are the time was a sister institute to some of the institutes affiliated with the J. Craig Venter Institute (JCVI) (it was a complicated time). Craig had led a project to do a massive amount of shotgun sequencing of DNA isolated from the Sargasso Sea, which had been the site of many previous studies of uncultured microbes. And Craig, as well as some of the people working with him including John Heidelberg who was at TIGR, had asked me to help in analysis of the data. So I eventually went to a meeting about the project and got involved. It was quite exciting and I put a lot of effort into helping analyze the data.

    As part of my work on the project, I and Martin Wu and Dongying Wu did a variety of phylogenetic studies of genes and gene families. One of these, was a phylogenetic analysis of proteorhodopsin homologs showing massively more diversity in the Sargasso data than in the PCR experiments done by Delong and Beja and others.

    Figure 7 from Venter et al. 2004. 

    We also did the first “phylotyping” in metagenomic data using genes other than rRNA. We built trees of bacterial ss-rRNAs, RecAs, RpoBs, HSP70s, EF-Tus and EF-Gs and then assigned each sequence to a phylum from the trees. In this analysis we found a variety of interesting things. 

    Figure 6 from Venter et al. 2004. 
    One thing I did not include in the Sargasso paper was an analysis I did of RecA homologs where I tried to include ALL RecA-like genes from bacteria, archaea, eukaryotes and viruses. The trees I made were a bit unusual but I was not sure that the alignments I had made were robust or that I had found all the RecA-like genes of interest so I did not even show this to Craig et al. at the time.
    UPDATE: I note – our work on this project was supported by a grant from the NSF Assembling the Tree of Life program that was awarded to me and Naomi Ward and Karen Nelson. Those funds supported the development of many of the informatics tools we used in this analysis and Martin and Dongying were both working on that project.

    After the Sargasso paper was published in 2004 though, I continued to fester about the RecA trees. And I wondered – if instead of trying to classify bacterial sequences into phyla, what if I tried to look for RecAs, rRNAs and other genes that were completely new branches in the tree of life? I got the chance to start to play with this concept again when Venter and crew asked me to help analyze the data coming out of the Global Ocean Sampling project. Again, this project was very exciting and interesting.

    As part of the project, I helped Shibu Yooseph and others look into whether the GOS data revealed any completely new types of functionally interesting genes, much like I had shown for proteorhodopsin in the Sargasso data.  

    Figure 7 from Yooseph et al. 2007 . Phylogenies Illustrating the Diversity Added by GOS Data to Known Families That We Examined 

    And again my mind started wandering towards the question of “OK – so – if there are all these very unusual and novel functionally interesting genes, what about looking for unusual and very novel phylogenetic marker genes”? So finally, I got back to work on the issue.

    And so I built a better RecA tree by first pulling out all possible homologs of RecA and RecA like proteins from the GOS data and then building an alignment and a tree. And there they were. Some very f*%&$ novel RecAs – distinct from any previously known RecA like proteins as far as I could tell. And so with help from Dongying and the JCVI crew, we started building a story about novel RecAs. And then we looked at RpoBs. And found novel ones too. And in mid 2006 while Shibu and Doug worked on their papers that were to be submitted to PLoS Biology and I worked on a review paper too, I told Emma Hill (who has since changed her name to Emma Ganley due to some sort of wedding thing) at PLoS Biology about the an analysis that was consistent with the existence of a fourth domain of life. No overstating our findings really – just that we found very novel phylogenetic marker genes. And that I was working on a paper on it. But alas I never got it done, though I was happy to have convinced Venter to send the GOS papers to PLoS Biology and I think the papers that came out were good. Among the papers were my review (Environmental Shotgun Sequencing: Its Potential and Challenges for Studying the Hidden World of Microbes, Doug Rusch’s diversity paper The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific and Shibu’s protein family paper The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families as well as many others as part of the Ocean Metagenomics Collection at PLoS.

    And in the midst of all of this, we had our first child and we wanted to move back to Northern California to be closer to family (my wife’s family is all in the Bay Area and my sister and brother Michael were in N. Cal too). So I applied for jobs and eventually took at job at UC Davis and we moved to Davis. Needless to say, all of that put a bit of a crimp in my work productivity. And once I was up and running at Davis, it just took a long time to get back to the searching for novel deep branches in the tree of life. But finally, we did it (with periodic prodding from Craig Venter). And we put together a paper and got it submitted to PLoS One in October. The reviews were very positive and enormously helpful. And we finally got a revision in January and it was officially accepted in February 2011. Only some seven years after my first work on the project. Whew.

    More detail of what is in the paper
    Well, I am going to be posting here some additional detail on what is in the paper.

    Why we punted on analysis of very novel rRNAs.

    The problem with rRNA is that the sequences that come from environmental samples are not complete (i.e. they only correspond to portions of the rRNA genes). Unfortunately, this makes a key step in phylogenetic analysis difficult – the alignment of sequences. We actually found about 200 rRNA sequences that seemed unusual in a phylogenetic sense. However, we were not convinced that the alignments of these fragments to other rRNAs was robust. This is because the alignment of rRNAs is best done making use of the base pairing secondary structure of the molecule and not the base sequence (i.e., primary structure).

    With only rRNA fragments, we could not use the secondary structure to do the alignments because you need to whole molecule to determine the best folding. Combined with the fact that we were searching for very distantly related ribosomal RNAs which would be hard to align even if we had the whole molecule, we were stuck for a bit. It seemed impossible to look for really novel organisms.
    So that is when we turned to other genes. The key for this is that there are protein coding genes that are universal and that for known organisms show similar patterns to rRNA in trees. In fact, in 1995 I wrote a paper showing that trees of RecA were very similar to trees of rRNA. RpoB is also considered a very robust phylogenetic marker. For organisms that we have in the lab (i.e., cultured) – many people use these other genes for phylogenetic analysis. rRNA has been very important in part because of the ease with which one can PCR amplify it from environmental samples and the fact that it is very hard to PCR amplify protein coding genes from the environment. Metagenomics changes this. With random sequencing, you get data from all genes. This means we can pick and choose genes to analyze for phylogenetic analysis and do not have to rely on rRNA.

    So we went after RecA first, because it has been shown to be a good phylogenetic marker for studies of the tree of life. And we found some very novel branches in the RecA tree. And after analyzing these and convincing ourselves that they were indeed phylogenetically very novel we went after RpoB. And also found very novel branches.

    So the phylogenetic analysis I think is very robust.

    RecA and RpoB as phylogenetic markers

    Many genes have been used as alternatives to rRNA genes to build “Trees of Life” including all organisms. Each has their own flavors of advantages and drawbacks. Two commonly used ones are the RecA and RpoB superfamilies.

    The many possible explanations for finding novel forms of phylogenetic marker genes

    The phylogenetically novel phylogenetic marker genes we found could have many explanations including that they could be ancient paralogs of these genes (but not found in any genomes we have available), they could be from viruses, or they could be from a novel branch on the tree of life. Or our trees could be bad. We think the latter is somewhat unlikely as our analysis has many lines of support. For example our RecA trees are very similar to those from a comprehensive study from M. Nei’s lab except they did not include the metagenomic data. But I guess it is still a possibility that our trees are biased in some way (e.g., by long branch attraction or bad alignments)

    Follow up analysis and rapid posting via Google Knol

    Amazingly and a bit sadly, I think we rushed the paper out. We left out one thing partly by accident – we had done an analysis of the locations from which these novel RecA and RpoB sequences had come. And somehow, in our final push to get the paper out, we left this out. I will be posting this information as soon as possible here and on the PLoS One site.

    In addition, after submitting the revision of our paper, we realized that we might be able to do a deeper analysis on one aspect of the work – how RpoB homologs from unusual DNA viruses compared to our novel sequences. We had included some RpoBs from DNA viruses in our analyses but not all that were available. So Dongying Wu did a very rapid additional analysis, adding some additional RpoB homologs to our alignment and making a tree of them. We then wrote a Google Knol about this new tree and submitted the Knol to PLoS Currents “Tree of Life” where it is currently in review. We are publishing the preprint of this Knol to make it available to all even while it is in review.

    Figure 2 from Wu and Eisen submitted. 

    Data availability

    There is a move afoot to make sure all data/tools associated with publications are readily available. We used publicly available sequence data and as much as possible publicly available tools for our work . We are trying to release as much as possible to allow people to re-analyze our work and to do any of the work themselves. We have therefore made use of the Dryad Data deposition service to post some of this material (see

    Who was involved

    • Dongying Wu a brilliant “Project Scientist” in my lab led the project (Project Scientist is one of the UC positions that is like what others call “Senior Scientist”). Dongying is simply one of the best bioinformaticians/computational biologists I have ever met. He was first author on many key papers from my lab including the Genomic Encyclopedia paper that came out last year and the glassy winged sharpshooter symbionts paper that came out a few years ago. Dongying worked in my group at TIGR and moved with me to UC Davis and currently splits his time between UC Davis and the DOE Joint Genome Institute. 
    • Martin Wu. Martin is an Assistant Professor at the University of Virginia. Prior to that he was a Project Scientist in my lab at Davis and a post-doc in my lab at TIGR. He is also a phenomenal bioinformatician / computational biologist. He developed the AMPHORA software in my lab and also led many genome projects (back when sequencing a genome was hard …) including that of the first Wolbachia genome and that of a very unusual bug Carboxydothermus hydrogenoformans. Martin helped with some of the genome analyses as part of this work. 
    • Aaron Halpern, Doug Rusch and Shibu Yooseph are all bioinformaticians from the J. Craig Venter Institute (Aaron is no longer there). All three helped with different aspects of dealing with and analyzing the GOS data and all three have been remarkably patient as this work dragged on and on. 
    • Marv Frazier from the JCVI was helpful in the initial set up and conceptualization of the project. 
    • J. Craig Venter is, well, Craig Venter, and he was involved in multiple aspects of the project including thinking about how and where to look for unusual sequences and interpreting some of the results.

    UPDATE: Funding for this work

    Most of my labs early work on this project was supported by a grant we had from the Assembling the Tree of Life program at the National Science Foundation (grant 0228651 to me and Naomi Ward). In that project we were working on sequencing and analyzing genomes from phyla of bacteria for which genomes were not available at the time. As part of this work we were designing methods to build phylogenetic trees from metagenomic data because we thought that our new genomes would be very useful in helping analyze metagenomic reads and figure out from which phyla they came. Later work on the project was supported by a grant to me, Jessica Green and Katie Pollard from the Gordon and Betty Moore Foundation (grant 1660).

    Some questions that might be asked and some answers (based in part on questions I have gotten from reporters). Note if you have other questions please post them here or on the PLOS One site for the paper.

    • Why no press release? Well, in part, because I sent information too late (shocking I know) to the Davis Press Office. But also because they have gotten suddenly busy with some Japan earthquake related actions. But also because, well, I really hate a lot of press releases. And finally, my brother had dinner with Carl Zimmer recently and apparently they discussed the possibility of having no press releases associated with papers. So here goes …. 
    • Really – what took so long? I would like to say the US Government made us hold back on publishing this until they could look into whether Venter collected ocean data from Roswell, NM or not. But really, the story above is true. We just did not get it done earlier. 
    • Why do you not know the source of the DNA (i.e., cells, viruses, etc)? This is why there was a six year wait between discovery and writing this up. We kept thinking we would be able to find the organisms but since I moved from TIGR and started a new job, we just never got around to getting to the source. We therefore decided to open this up to others who will hunt for the source by writing up the paper. 
    • Why did you not rename the Unknown 2 group in the RecA tree? We should have renamed our group “Thaumarchaeota” or something like that. When we did the initial analysis our group was novel. And then a few years ago a few groups obtained data from what is thought to be the third major lineage of Archaea – referred to by some as Thaumarchaeota. This is to go with the Euryarchaeota and Crenarchaeota. See for example. 
    • One of the clades in the RecA tree (XRCC2) seems out of place phylogenetically. I can see how that is confusing. The XRCC2 clade is very weird and hard to figure out. It is not the “normal” eukaryotic genes – those are the Rad51/DMC1 genes. One complication with the RecA family is that there have been duplication events to go with the species evolution. And thus eukaryotes have Rad51, DMC1, Rad51B, Rad51C, Rad57, XRCC3 and XRCC2. We tried to figure out where the XRCC2 group should go but it just was hard to place. The statistical support for its position (we used a method called bootstrapping) is low (note the lack of a number on the node where the branch leading to XRCC2 connects to the base of the tree). Most likely that group should be placed with some of the other eukaryotic groups. However, it seems likely that there was a duplication in the lineage leading up to the ancestor of eukaryotes and archaea (some studies have indicated they share a common ancestor to the exclusion of bacteria). Such a duplication would explain why basically all archaea have a RadA and and RadB and all / most eukaryotes have multiple paralogs as well. 
    • The Unknown 1 group in the RpoB RecA tree seems to group with phage. What can you say about that? We think unknown 1 is potentially of viral origin but still cannot tell. The fact that is clusters with RecA superfamily members from phage suggests this but it is distant enough from known phage for us to not be confident in any predicted origin. As for derivative forms vs. independent branch – this is one of the big questions about viruses these days. Many viruses encode homologs of “housekeeping” genes found across bacteria, archaea and eukaryotes. And in many cases the viral versions of these genes appear to phylogenetically very novel. This is why the people studying mimivirus (which we refer to) suggest some viruses may in fact represent a fourth branch on the tree of life. It is possible that some viruses are in fact reduced forms of what were once cellular organisms – akin to parasitic intracellular species of bacteria possibly. 
    • Why are these phylogenetically novel sequences so low in abundance? This is a key question. I think it would be easy to come up with a theory for these being rare or these being common. They might be rare if their niche is very limited today. Or they might be rare because they could not be very competitive with other organisms. Or they could be rare because they require some unusual interactions with other taxa. In addition, we have only looked carefully at ocean water samples. If these are common somewhere else (e.g., hotsprings, deep subsurface, etc) we would not yet have figured that out. We are looking at additional metagenomic data right now to see fi we can find any locations where relatives of these genes are more common

    Some related papers by others worth looking at

    Some related papers by me possibly worth looking at

    Some related blog posts I have written over the years

      Dongying Wu, Martin Wu, Aaron Halpern, Douglas B. Rusch, Shibu Yooseph, Marvin Frazier,, & J. Craig Venter, Jonathan A. Eisen (2011). Stalking the Fourth Domain in Metagenomic Data: Searching for, Discovering, and Interpreting Novel, Deep Branches in Marker Gene Phylogenetic Trees PLoS One, 6 (3) : 10.1371/journal.pone.0018011