New paper: PLoS ONE: Accounting For Alignment Uncertainty in Phylogenomics

A new paper is out from the lab: PLoS ONE: Accounting For Alignment Uncertainty in Phylogenomics.

It describes the “Zorro” software for automated alignment masking.


Uncertainty in multiple sequence alignments has a large impact on phylogenetic analyses. Little has been done to evaluate the quality of individual positions in protein sequence alignments, which directly impact the accuracy of phylogenetic trees. Here we describe ZORRO, a probabilistic masking program that accounts for alignment uncertainty by assigning confidence scores to each alignment position. Using the BALIBASE database and in simulation studies, we demonstrate that masking by ZORRO significantly reduces the alignment uncertainty and improves the tree accuracy.

This work was supported by the Gordon and Betty Moore Foundation.

New publication from the lab: Assemblathon 1: A competitive assessment of de novo short read assembly methods

Aaron Darling from the lab is an author on a new paper just published: Assemblathon 1: A competitive assessment of de novo short read assembly methods.

New paper from my lab (& the Facciotti lab): Mauve Assembly Metrics #Halophiles #Genomics

Just a quick post here. A new paper from my lab has come out in Bioinformatics. The paper is relatively simple. Titled “Mauve Assembly Metrics” it reports work of Aaron Darling and Andrew Tritt (with some minor contributions from me and Marc Facciotti). Aaron wrote the program Mauve when he was a student in Nicole Perna’s lab at Wisconsin: Mauve: multiple alignment of conserved genomic sequence with rearrangements. Over the years he (and others) have continued to develop the program and written a few papers too including for example, the development of progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. This new paper reports basically a system/scripts to measure assembly quality. Here is the abstract:

High throughput DNA sequencing technologies have spurred the development of numerous novel methods for genome assembly. With few exceptions, these algorithms are heuristic and require one or more parameters to be manually set by the user. One approach to parameter tuning involves assembling data from an organism with an available high quality reference genome, and measuring assembly accuracy using some metrics. We developed a system to measure assembly quality under several scoring metrics, and to compare assembly quality across a variety of assemblers, sequence data types, and parameter choices. When used in conjunction with training data such as a high quality reference genome and sequence reads from the same organism, our program can be used to manually identify an optimal sequencing and assembly strategy for de novo sequencing of related organisms.

Check out the paper: Mauve Assembly Metrics. Download the scripts/code and Mauve and play around and let me know what you think.
Note this paper was supported by a grant from the National Science Foundation (ER 0949453). That grant is focused on comparative genomics (sequencing and analysis) of halophlic archaea. Stay tuned for more on that project as we are writing up a series of papers ….
Some related links:

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

      More coverage of the GEBA "Phylogeny Driven Genomic Encyclopedia"

      Just a quick note here to post some links to additional stories about my new paper on “A phylogeny driven genomic encyclopedia of bacteria and archaea” which was published last week in Nature (with a Creative Commons license – which is rare in Nature but is what they use for genome sequencing papers).

      Carl Zimmer has an article today in the New York Times “Scientists Start a Genomic Catalog of Earth’s Abundant Microbes”  about the paper and the project.  In the article he interviews me and Hans-Peter Klenk, who was a co-author and led the culturing part of the project.  A few things to note about this:

      • It is rare to have archaea mentioned in the New York Times.
      • There is a tree that goes along with the article which is a modified version of the tree we had in our paper.  I think theirs is very nice. Kudos to their artist
      • There is a quote by Norm Pace generally supportive of the project 
      • The article mentions the JGI Adopt a Microbe program and even has a shout out to Malcolm Campbell at Davidson College and his recent PLoS One paper where they discuss results from a project where they took one of the genomes from our project and used it as part of a course on genome annotation/analysis. 

      For some of the story behind the paper see my recent blog post “Story Behind the Nature Paper on ‘A phylogeny driven genomic encyclopedia of bacteria & archaea’ #genomics #evolution

      Other discussions worth checking out

      Also see

      Wu, D., Hugenholtz, P., Mavromatis, K., Pukall, R., Dalin, E., Ivanova, N., Kunin, V., Goodwin, L., Wu, M., Tindall, B., Hooper, S., Pati, A., Lykidis, A., Spring, S., Anderson, I., D’haeseleer, P., Zemla, A., Singer, M., Lapidus, A., Nolan, M., Copeland, A., Han, C., Chen, F., Cheng, J., Lucas, S., Kerfeld, C., Lang, E., Gronow, S., Chain, P., Bruce, D., Rubin, E., Kyrpides, N., Klenk, H., & Eisen, J. (2009). A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea Nature, 462 (7276), 1056-1060 DOI: 10.1038/nature08656

      Bakke, P., Carney, N., DeLoache, W., Gearing, M., Ingvorsen, K., Lotz, M., McNair, J., Penumetcha, P., Simpson, S., Voss, L., Win, M., Heyer, L., & Campbell, A. (2009). Evaluation of Three Automated Genome Annotations for Halorhabdus utahensis PLoS ONE, 4 (7) DOI: 10.1371/journal.pone.0006291

      Story Behind the Nature Paper on ‘A phylogeny driven genomic encyclopedia of bacteria & archaea’ #genomics #evolution

      Today is a fun day for me. A paper on which I am the senior author is being published in Nature (yes, the Academic Editor in Chief of PLoS Biology is publishing a paper in Nature, more on that below ..). This paper, entitled, “A phylogeny driven genomic encyclopedia of bacteria and archaea” represents a culmination of years of work by many people from multiple institutions. Today in this blog I am going to do my best to tell the story behind the paper – about the people and the process and a little bit about the science.

      First, a brief bit about the science in the paper. In this paper, we (mostly people at the Joint Genome Institute, where I have an Adjunct Appointment — but also people in my lab at UC Davis and at the DSMZ culture collection) did a relatively simple thing – we started with the rRNA tree of life as a guide. Then we identified branches in the bacterial and archaeal portions of this tree where there were no genome sequences available (or in progress) (this was done mostly by Phil Hugenholtz, Dongying Wu and Nikos Kyrpides) Next we searched for representatives of these “unsequenced” branches in the DSMZ culture collection (a collection of bacteria and archaea that can be grown in the lab). And we identified in total some 200 of these. And then the DSMZ (under the direction of Hans-Peter Klenk) grew these organisms and sent the DNA to the Joint Genome Institute. And then JGI turned on their genome sequencing muscle and sequenced the genomes of the organisms in the DNA samples. And finally, we spent a good deal of time then analyzing the data asking a pretty simple question – are there any general benefits that come from this “phylogeny driven” approach to sequencing genomes compared to what one might find with sequencing just any random genome (after all, any genome sequence could have some value)? The paper, describes what we found, which is that there are in fact many benefits that come from sequencing genomes from branches in the tree for which genomes are not available.

      More on the details of the science below. But first, I want to note that this paper was truly an amazing team effort, with all sorts of people from the JGI in particular, going above and beyond the call of duty to make sure it happened and worked well. And the Department of Energy has been truly phenomenal in my opinion in supporting this project which in the end is not explicitly about “energy” per se but is really about providing a reference set of genomes that should improve the value of all microbial genome data.

      Anyway, now for the story behind the story. And be prepared, because this is a bit long. But I think it is important to place this work in a bigger context both in terms of my background as well as some of the background of other people in the project. If you can’t wait for more on the GEBA project then perhaps you should go to some of these links:

      And I will post more links as they come up. Below what I try to provide is some of the story behind the story:

      My personal interest in applied uses of phylogenetics stage 1: undergraduate preparation at Harvard
      As this paper is primarily about an applied use of phylogenetics (in selecting genomes for sequencing), I thought it would be worth going into some of how I personally became a bit obsessed with applied uses of phylogenetics. For me, my obsession began as an undergraduate at Harvard where I got exposed to the value of phylogeny as a tool from many many angles including but not limited to:

      • Freshman year taking a course taught by Stephen Jay Gould where Wayne and David Maddison were Teaching Assistant’s and where they were demoing their new phylogenetics software called MacClade
      • Sophomore year taking a conservation biology class with Eric Fajer and Scott Melvin where I was exposed to the concept of “phylogenetic diversity” as a tool in assessing conservation plans
      • Junior year working in the lab of Fakhri Bazzaz with people like David Ackerly and Peter Wayne who made use of phylogeny as a key tool in their research projects
      • Senior year and the year after graduating where I worked in the lab of Colleen Cavanaugh using rRNA based phylogenetic analysis to characterize uncultured chemosynthetic symbionts. I note it was in Colleen’s lab that I also became obsessed you could say with microbes and why they rock.
      My personal interest in applied uses of phylogenetics stage 2: graduate school at Stanford
      All of this and more gave me a strong passion for phylogeny as a tool. And so when I went to graduate school at Stanford (originally to work with Ward Watt on butterflies, but then I switched to working in Phil Hanawalt‘s lab on the “Evolution of DNA repair genes, proteins and processes“). And while in that lab I become pretty much obsessed with three things, all related to phylogeny.
      First, I was interested in whether the rRNA tree of life, which I had used in my studies in Colleen Cavanaugh’s lab (and in my first paper in J. Bacteriology, which, thanks to ASM, is now in Pubmed Central and free at ASM’s site too), was robust or, as some critics argued, was not that useful. This was a critical question since the best way to study the phylogeny of microbes at the time, and also the best way to study uncultured microbes, was to leverage the ability to clone rRNA genes by PCR and then to build evolutionary trees of those rRNA genes. As part of my graduate work, I did a study where I compared the phylogenetic trees of rRNA to trees of another gene from the same species (I chose, recA). Surprisingly, despite the claims that the rRNA tree was not very useful and that different genes always gave different trees, if you compared the two trees ONLY where there was strong support for a particular branching pattern, the trees of the two genes were in fact VERY VERY similar (a finding that had been suggested previously by others, including Lloyd and Sharp)
      Second, I also became obsessed with the fact that most of the experimental studies of DNA repair processes were in a very narrow sampling of the phylogenetic diversity of organisms (e.g., at the time, no studies had been done in archaea, and most studies in bacteria were from only two of the many major groups). So I started experimental studies of repair in halophilic archaea in order to help broaden the diversity of studies. And I began to use PCR to try and clone out repair genes from various other species of diverse bacteria and archaea. Alas, as I was doing this, some institute called TIGR was sequencing the complete genomes of organisms I was trying to clone out single genes from. In fact, one of the first organisms I was working on for PCR studies was Archaeoglobus fulgidus. And when I found out TIGR was sequencing the genome, in a project led by non other than the great microbial evolutionary biologist Hans-Peter Klenk (yes, the same one who helped us in this GEBA project). I decided it was silly to try to clone out individual genes by PCR. And thus I began to learn how to analyze genomes.
      It was in the course of learning how to analyze genomes that I came up with another applied use of phylogeny. I realized that one should be able to use phylogenetic studies of genes to help in predicting functions for uncharacterized genes as part of genome annotation efforts. And so I wrote a series of papers showing that this in fact worked (I did this first for the SNF2 family of proteins and then alas coined my own omics word “phylogenomics” to describe this integration of genome analysis and phylogenetics and formalized this phylogenomic approach to functional prediction). I note that what I was arguing for was that protein function could be treated like ANY other character trait and one could use character trait reconstruction methods (which I had learned about while playing with that MacClade program) to infer protein functions for unknown proteins in a protein tree. I note that this notion of predicting protein function from a protein tree is completely analogous to (and one could rightfully say stolen from) how researchers studying uncultured microbes were trying to infer properties of microbes from the position of their rRNA genes in the rRNA tree of life (as I had learned in studies of symbioses).
      My personal interest in applied uses of phylogenetics stage 3: my plans for a post doc
      So as I was wrapping up graduate school I was seeking a way to go beyond what I was doing and combine studies of DNA repair and evolution and microbiology in another way. And I thought I had found a perfect one in a post doc I accepted with A. John Clark at U. C. Berkeley. John was the person who had discovered recA, the gene I had been using for phylogenetic analysis and for structure function studies. And he was working with none other than Norm Pace and a young hotshot in Norm’s lab, Phil Hugenholtz (as well as a few others including Steve Sandler) in trying to use the recA homolog in archaea as a marker for environmental studies of archaea. It sounded literally perfect. And so I was excited to start this job. That was, until I met Craig Venter.
      Grabbing the TIGR by the tail
      While I had been playing around with data from TIGR in the latter years of my time in graduate school, I also got involved in teaching a fascinating class with David Botstein, Rick Myers, David Cox and others. (As an aside, this class was part of a new initiative I helped design at Stanford on “Science, Math and Engineering” for non science majors – an initiative that was a pet project of non other than Condie Rice who was Provost at the time). Anyway, Rick Myers was serving as a host for one Craig Venter when he came and gave a talk at Stanford and somehow I managed to finagle my way into being invited to go out to dinner with Craig. And at dinner, I proceeded to tell Craig that I thought some of the evolution stuff he was talking about was bogus and I tried to explain some of my work on phylogeny and phylogenomics. Not sure what Craig thought of the cocky PhD student drawing evolutionary trees on napkins, but it eventually got me a faculty job at TIGR and I worked extensively with Craig so it must have been worth something. And so I and my fiancé Maria-Inés Benito (now wife …) moved to Maryland and spent eight great years there (my wife started in MD as a faculty member at TIGR too, but then she left to go to a company called Informax, may it rest in peace).

      Most of my work at TIGR focused on a different side of phylogenomics than represented in the GEBA project. At TIGR I focused on the uses of evolutionary analysis as a component to analyzing genomes – from predicting gene function to finding duplications (e.g., see the V. cholerae genome paper) to identifying genes under unusual patterns of mutation or selection to finding organelle derived genes in nuclear genomes (e.g., see this) to studying the occurrence of lateral gene transfer or the lack of occurrence of it to studying genome rearrangement processes.. And sure, every once in a while I worked on a project where the organism was the first in its major branch to have a genome sequenced (e.g., Chlorobi). And I had noted, along with others that there was a big phylogenetic bias in genome sequencing project (e.g., see my 2000 review paper discussing this here).

      But that did not really drive my thinking about what genome to actually sequence until TIGR hired a brilliant microbial systematics expert Naomi Ward as a new faculty member. And it was Naomi who kept emphasizing that someone should go about targeting the “undersequenced” groups in the Tree of Life.

      NSF Assembling the Tree of Life grant
      And so Naomi and I (w/ Karen Nelson and Frank Robb) put together a grant for the NSF’s “Assembling the Tree of Life” program to do just this – to sequence the first genomes from eight phyla of bacteria for which no genomes were available but for which there were cultured organisms. Amazingly we got the grant. And we did some pretty cool things on that project, including sequencing some interesting genomes, and developing some useful new tools for analyzing genomes (e.g., STAP, AMPHORA, APIS). And I was able to hire some amazing scientists to work in my lab on the project including Dongying Wu (the lead author on the GEBA paper) and Martin Wu (who also worked on the GEBA project and is now a Prof. at U. Virginia) and Jonathan Badger. Alas, we did not publish any earth shattering papers as part of this NSF Tree of Life project on analyzing the genomes of these eight organisms, not because there was not some interesting stuff there but for some other reasons. First, I moved to UC Davis and there was a complicated administrative nightmare in transferring money and getting things up and running at Davis on this project so my lab was not really able to work on it for two years (in retrospect, what a f*ING nightmare dealing with the UC Davis grants system was …).

      Then, just as things we ready to get restarted, TIGR kind of imploded and many of the people, including Naomi, my CoPI, left (though I note, my moving to Davis was unrelated to the dissolution of TIGR). But perhaps most importantly, there were some actual technical and scientific problems with our dreams of changing the world of microbiology from our phyla sampling project – the science was not quite there. In particular, having a single genome from each of these phyla was simply not enough to get (and show) the benefits that can come from improved sampling of the tree of life. And thus though we have published some cool papers from this project (e.g., see this PLoS One paper on one of the genomes), we all ended up in one way or another, disappointed with the final results.

      Davis and JGI: the return of phylogeny to genomic sampling
      When I moved to UC Davis I also was offered (and accepted) an Adjunct Appointment at the Joint Genome Institute (JGI). At both places, I envisioned reinventing myself as someone who worked on studying microbes directly in the environment (e.g., with metagenomics) and symbioses (both of which I had started on at TIGR). And in fact, in a way, I have done this, since I got some medium to big grants to work on these issues. I tried diligently to attend weekly meetings at the JGI but it was difficult since technically I was 100% time at UC Davis and was in essence supposed to be at 0% time at JGI. And when JGI hired Phil Hugenholtz to run their environmental genomics/metagenomics work, I was needed less at JGI since, well, Phil was so good. It was great to go over there and interact with Eddy Rubin, Phil Hugenholtz, and Nikos Kyrpides, among others, but it was unclear what exactly I would do there with Phil running the metagenomics show.

      And then, like magic, something came up. I went to one of the monthly senior staff meetings at JGI and while we were listening to someone on the speaker phone, Eddy Rubin handed me a note asking me what I thought about the proposal someone was making to sequence all the species in the Bergey’s Manual. And the light bulb of phylogeny went back on in my head. I told him (I think I wrote it down, but may have said out loud), something like “well, sequencing all 6000 or so species would be great, but it would be better to focus on the most phylogenetically novel ones first.” And in a way, GEBA was born. Eddy organized some meetings at JGI to discuss the Bergey’s proposal and I argued for a more phylogeny driven approach. And this is where having Phil Hugenholtz and Nikos Kyrpides at JGI was like a perfect storm. You see, both had been lamenting the limited phylogenetic coverage of genomes for years, just like I had. Phil had even written a paper about it in 2002 which we used as part of our NSF Tree of Life proposal. And Nikos too had been diligently working for years to make sure novel organisms were sequenced. So though we went to a meeting to discuss the Bergey’s manual idea, we instead proposed an alternative – GEBA.

      And for some months, we pitched this notion to various people including at JGI, DOE, and various advisory boards. And the response was basically – “OK – sounds like it COULD be worth doing – why don’t you do a pilot and TEST if it is worth doing” And so, with support from Eddy Rubin and DOE, that is what we did.

      One key limitation – getting DNA

      So Phil, Nikos and I and a variety of others starting working on the general plan behind GEBA. But there was one key limitation. How were we going to get DNA from all these organisms? One possibility was to seek out diverse people in the community and have them somehow help us. This had some serious problems associated with it, not the least of which was the worry that we might end up sequencing varieties of organisms that people had in their lab but which nobody else had access to (something Naomi Ward and I had written about as a problem a few years before).

      And here came the second perfect storm – none other than Hans-Peter Klenk (yes, the same one who had led some of the early genome sequencing efforts when he was at TIGR), was visiting JGI. And he had a relatively new job – at the German Culture Collection DSMZ (In fact, I should note, I had tried to get a job at TIGR even before I met Venter, since they had a position advertised for a “microbial evolutionary biologist” — but that job went to Klenk). Phil Hugenholtz had asked the Head of DSMZ, Erko Stackebrandt, if they might be interested in helping us grow strains and get DNA but we did not yet have a full collaboration with them. And Erko had suggested we contact Hans-Peter. And in his visit to JGI it became apparent that he would do whatever he could to help us build a collaboration with DSMZ. And thus we had a source of DNA. Even more amazingly to me, they did it all for free.

      GEBA begins

      And thus began the real work in the project. Phil used his expertise with rRNA databases, especially GreenGenes, to pull out phylogenetic trees of different groups. And Nikos used his expertise as the curator of a database on microbial sequencing projects (called GenomesOnline) to help tag which branches in Phil’s tree had sequenced genomes or ones in progress. And then they looked for whether any of the members of the unsequenced branches had representatives in the DSMZ collection. And with some help from Dongying Wu and me, we came up with a list. And with the help of the JGI “Project Management” team including David Bruce and Lynne Goodwin and Eileen Dalin and others at JGI we developed a protocol for collaborating with DSMZ and getting DNA from them.

      And I became the chief cheerleader and administrator of the project, in part since Phil and Nikos were so busy with their other things at JGI. And though I was not always on the ball, the project moved forward and we started to get genomes sequenced using the full strength of the JGI as a genome center. The finishing teams at JGI worked diligently on finishing as many of the genomes as possible. And Nikos’ team at JGI made sure that the genomes were annotated. And I helped make sure that they data release policies were broadly open (which everyone at JGI supported). And after many false starts with papers on the project that were way way way to cumbersome and big, with some kicks in the pants from the director of JGI Eddy Rubin who was getting anxious about the project, we turned out the GEBA paper that was published today in Nature.

      You might ask, why, as a PLoS official and PLoS cheerleader, we ended up having a paper in Nature? Well, in the end, though I am senior author on the paper, the total contribution to the work mostly came from people at JGI who did not work for me but instead worked with me on this great project. And we took some votes and had some discussions and in the end, despite my lobbying to send it to PLoS Biology, submitting it to Nature was the group decision. I supported this decision in part due to the fact that Nature uses a Creative Commons license for genome papers. But I also supported it because in the end, this was a collaboration involving many many many people and in such projects everyone has to compromise here and there. Now mind you, I am not sad to have a paper in Nature. But I would personally have preferred to have it in a journal that was fully open access, not just occasionally open like Nature.

      Now I note, there were a million other things that went on associated with the GEBA project. Some of which I was not even involved in in any way. I will try to write about some of these another time, but this post is already way way way too long. So I am going to just stop here and add that I have been honored and lucky work with people like Phil, Nikos, Hans-Peter, and others on this project and to have the people at the JGI work so hard on the background parts of this project. Thanks to all of them and to the people at DSMZ and in my lab who helped out and to the DOE for funding this work (as well as the Gordon and Betty Moore Foundation, who funded some of the work from my lab on analysis of these genomes). And last but not least, thanks to the Director of JGI Eddy Rubin, supporting this project and for being patient with it and for kicking us in the pants when we needed to get moving on getting a paper out.

      Wu, D., Hugenholtz, P., Mavromatis, K., Pukall, R., Dalin, E., Ivanova, N., Kunin, V., Goodwin, L., Wu, M., Tindall, B., Hooper, S., Pati, A., Lykidis, A., Spring, S., Anderson, I., D’haeseleer, P., Zemla, A., Singer, M., Lapidus, A., Nolan, M., Copeland, A., Han, C., Chen, F., Cheng, J., Lucas, S., Kerfeld, C., Lang, E., Gronow, S., Chain, P., Bruce, D., Rubin, E., Kyrpides, N., Klenk, H., & Eisen, J. (2009). A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea Nature, 462 (7276), 1056-1060 DOI: 10.1038/nature08656

      Tackling a Hairy Beast II

      For all out there who love ciliates and their relatives, you might want to check out the second paper to come out of my Tetrahymena thermophila Genome Sequencing Project for which the preprint is available in BMC Genomics.
      In this project we have been sequencing, annotating and finishing the macronuclear genome of this lovely organism. Like other ciliates Tetrahymena has two nuclei and two nuclear genomes – the macronucleus (MAC) and the micronucleus (MIC). The MIC is analogous to germ cells in animals — it is sort of a genomic repository for sexual reproduction. After sexual reproduction, the MIC genome is processed to generate the MAC genome which is then used in an analogous way to soma cells in animals (the MAC is the site for most/all gene expression in Tetrahymena). I have been the PI on this project which was supported by grants from NSF and NIH and was a collaboration involving TIGR (where I used to work), Stanford, UCSB, JCVI (which subsumed TIGR a few years ago) and the Tetrahymena research community.
      Our first paper on this project was published in PLoS Biology two years ago. I have written about it previously here.

      The new paper describes further work on the MAC genome including finishing many of the chromosomes (which was done spectacularly by Luke Tallon and Kristie Jones), sequencing and analyzing a larger number of ESTs, refining the annotation (coordinated by Mathangi Thiagarajan), and some other analyses. The new paper was led by Bob Coyne, who, with Barb Methé took over coordinating the work at TIGR/JCVI after I moved to UC Davis a few years back. I think they did a stellar job (ni biases here).

      Note – I took the title of the posting ‘Tackling a Hairy Best” from an NIH press release that was put out when we got the grants for this project.