Thanks for writing!!! It’s so glad an actual TiGRfam guy has looked over the piece. I too wish that I could have met you at the session in person. Like everyone at the Venter Institute, you really know your stuff!

I figured when I wrote this article that I probably hadn’t gotten everything just right. My problems were a) trying to understand the material myself b) trying to explain it to others in simple terms.

That the TIGRfam is a match to an HMM..whoops! I think I got a basic understanding of what an equivalog is and was going to try to explain it and the cutoff point in the article but could not find the right words. So thank you for providing a concise and easy to understand description of how they are linked.

About TIGR…another whoops! I guess I had the actual tiger statue in mind when thinking about the Institute’s prior name. By the way, each day I took a shuttle from my hotel (the Sleep Inn) over to JCVI. When I told them I wanted to go to JCVI the shuttle drivers looked at me with a blank stare. When I gave them the address they got excited, repeating “Oh TIGR, TIGR! So you are a TIGER! Apparently they call people going to JCVI “tigers” based on the prior name. They will radio each other and say, “I’m headed to pick up five tigers!” Anyway, found that amusing but fear they need to be updated on the new name.

As for the closed and open genomes, I greatly appreciate your elegant and more accurate description of how they differ. I am currently writing a paper on deadline, but as soon as I get the chance I will amend the article according to your corrections.

I’m glad this piece found it’s way to you! If you see Tanja or any of my other teachers be sure to tell them I say hi …and keep up the fantastic work you guys are doing out there!

Best,

Amy

The predecessor to JCVI was TIGR (not Tiger) which stood for The Institute for Genomic Research. The “T” for The was included so that it didn’t come out like Igor!

Prakaryotes include Bacteria as well as Archaea which are now recognized as a separate third domain of life. The term isn’t really phylogenetically proper since it would appear that the Archaea are more closely related to Eukaryotes than the Bacteria. Nevertheless the term serves a useful purpose since Archaea and Bacteria share much in the way of basic lifestyle and, importantly for genomics, genomic organization (read, no introns (!) and lots of operons (!!)).

Since I’m one of the TIGRFAMs guys (sorry I missed you when you were here for your course, I usually stop in to help answer questions, but I’ll fill that role here, I guess!), I’ve got to chime in on your TIGRFAMs example. The “TIGRFAMs ID you refer to is a match to one of those HMMs you discussed, not a match to a single annotated protein. In making our TIGRFAMs models, we strive to model “equivalogs”, families of related proteins which all have the same function. We try and combine the sequences of not just one protein which has been well characterized, but as many as possible, and we also include other types of evidence that strengthen the case that all have the same function (like genomic context and the construction of evolutionary trees). Finally, we curate a “trusted” score. When your unknown sequence is evaluated by the HMM and a score is assigned, you don’t have to ask how high is high enough; if it’s above the trusted score you can have high confidence that it really has the same function as all the other members of that family.

Your comment about “closed” and “open” genomes missed the mark a little. Genomes are open if there was not enough DNA sequence collected to fill all the gaps and create a complete picture of every part of the genome at the level of the DNA, its order and orientation. Often the amount of DNA missing or potentially mis-positioned is quite small. Whether open or closed, genomes submitted to public databases are always “works in progress” with respect to functional annotation. A large number of genes annotated with our best efforts today will appear to be incomplete or incorrect next year. Coming up with systems to update all of these “stale” annotations as our base of knowledge increases is one of our primary challenges going forward.

Glad you enjoyed the article and thanks for your kind words.

Gene, thank you very much for referring me to the study Dr. Blaney posted on the MP site in which Shingella bacteria are implicated in endometriosis. It will be great to have that study on hand at Porto.

I see no reason why the techniques used by the researchers in the Indian study could not be used to identify the presence of certain forms of latent bacteria in the tissues of people with inflammatory disease.

However I know that Dr. Marshall’s technology of preference when it comes trying to detect chronic pathogens in the cells of patients with inflammatory disease is single cell sampling or shot-gun sequencing.

To be more specific, he would ideally like to use the same equipment and study design that Dave Relman of Stanford University used in the following study:

http://www.pnas.org/content/104/29/11889

I believe that Dr. Marshall already met several researchers at last year’s Metagenomics Conference who showed an interest in helping him use shotgun sequencing in order to detect the bacteria harbored by certain MP patients. For one thing, the project would cost more money than ARF currently has at its disposal. But according to Dr. Marshall even if ARF had the money, it wouldn’t really make sense to proceed with the project until the Human Microbiome Project has specifically named and identified more of the bacterial species involved in chronic disease. There’s not much point in doing the single cell sequencing right now and finding out that we have a lot of “unknown” sequences on our hands. They would likely correspond to bacterial genomes, yet we would have no real proof to make that assumption. The results might end up reflecting ambiguity which could possibly even hurt perception of the MP.

Marshall is also still very interested in the flourescent dye that Dr. Aguzzi discussed at Karolinska (read about the dye in my Prions article). He believes that the dye will cause bacterial polymers in the blood to flouresce. So the dye might prove to be the best way to detect the quantity of bacteria in a given sample. Then after more bacterial sequences are annotated by the Human Microbiome Project, performing shotgun sequencing on the same sample would be the best way to determine the quality of bacteria in the sample. By quality I mean what species are present and their level of pathogenicity.

If these two methods are used to analyze the blood samples of a patient before the MP then it can at least be expected that after several years on the treatment their blood would flouresce to a much lesser degree. As for the quality of the pathogens, the sample would likely not be bacteria free, but the DNA present would hopefully correspond to the DNA of more innocuous species.

I’m excited for such analysis to take place which is why I have a great interest in the Microbiome Project. I hope the scientists involved move forward with deliberate speed (as they are planning to!) so that we will have many more comparison sequences to work with as soon as possible.

Best,

Amy

WOW!!! What an excellent opportunity you had to visit the Craig Venter Institute and participate in a training session on genome sequencing. Great reporting!

I was interested in the human genome project at the time and it was amazing how Venter and Celera Genomics were able to accelerate the over all project. It is good to see he is still challenging the “status quo” by moving forward with new ideas.

Another interesting source for bacteria (rather than nose, grin would be the cells of people infected with CWD bacteria.

As you know, the cell mRNA which represents gene transcription, can be extracted at a “point in time” and converted to complementary DNA (cDNA) using reverse transcriptase, then amplified with polymerase chain reaction (PCR). Analysis by the JCVI would show if there is any cellular bacterial DNA confirming the MP. This is the technique used in the research study posted by Dr. Blaney on the MP site in regard to endometriosis being caused by infection.

Thinking ahead, once the bacterial genome is understood and the bacteria mRNA identified, it may be possible to block that mRNA from the bacteria 70S ribosome with Antisense RNA. The Antisense RNA hybridizes to, and makes the mRNA double stranded so it cannot pass into/through the ribosome. This may later be away to replace the MP abx and get better control of IP.

These are some of my thoughts. We certainly live in interesting times!!! Thanks to Dr. Marshall and the MP we are at the leading edge of this science.

Gene