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This is the BIOL375 class of 2010-11. These are the students currently working with Dr. Scott on the Meiothermus ruber genome annotation project. This presentation was created by students in this course.

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1MEIOTHERMUS RUBER

Mitch Anliker, Mohammed Hussain, Heather Smith, Melissa Reller, Jose Candelario OrozcoIntroduction

Background Information on Meiothermus ruberExplain what it means to "annotate"For what Purpose?Click on the speaker Icon to learn more about the Meiothermus ruber project and annotations

3My name is Melissa, and I am a biology major here at Augustana. My name is Heather, and I am a biology/pre-vet. major here at Augustana.My name is Jose and I am a biochemistry/ chemistry double major.

My name is Mitch, and I am biochemistry/premed major

In this presentation we are going to provide background information on Meiothermus Ruber, our bacteria of interest. In addition, we are going to explain what you are going to do, which is gene annotation.In lab, current students are amplifying the Meiothermus ruber genomic DNA using PCR reactions.

BACKGROUNDWhat is Meiothermus ruber? Procaryote: Eubacteria domain

Physical characteristics: Thermophile - prefers 120-140F Isolated from hot springs Pest in paper mills Non-pathogenic

Genome:3,000,000 base pairs *3,100 protein-coding genes predicted

4What is Meiothermus ruber? Let's begin with the fact that it is a bacteria or a procaryote and it is in the Eubacteria domain. It is characterized by the production of a red pigment. It is aerobic-meaning it requires oxygen to survive.Meiothermus ruber has not been researched on a lot but through it has been found that it is a thermophile. This means that it can live in hot temperatures and preferably it lives in 120-140 degrees F. Meiothermus ruber is primarily found in the hot springs. This bacteria is non-pathogenic which means we will not be able to catch it. As for how big this bacteria is, it has about 3 million base pairs and 3,100 protein-coding genes.This bacteria was a problem in paper mills because the solution they used was kept at a high temperature, therefore this bacteria invaded the paper mills. Studying this many genes is a lot for one person, so this is one of the many reasons why we need you to help us research and figure out more about Meiothermus ruber.BACKGROUND CONTINUEDPhylum: ThermiClass: ThermiOrder: ThermalesFamily: ThermaceaeGenus:MeiothermusSpecies: ruberPure science reasonsMost thermophiles belong to the Archaea domainDOEs GEBA projectUndergraduate research

5Another reason is to help out the DOE Joint Genome Institute with this research for possible energy and environmental benefits.One last reason would be to help give you a way to gain experience in research on something that has not been studied a lot. This gives you several possible outcomes just from the research you could do. Therefore, the next thing is to learn about this bacteria to use the programs that JGI set up for you, and look into the different genes in the bacteria. This process of using these programs will essentially give you an annotation of the gene.Why study Meiothermus ruber?Practical reasons Contaminant of paper millsContains an enzyme that digests feathers

6The practical reasons why Meiothermus ruber is being studied is to figure out a way to stop the invasion of this bacteria in paper mills and to use this bacteria safely as it contains an enzyme that breaks down feathers. Since this bacteria is non-pathogenic and can live at high temperatures, it was found that it is a good candidate to break down feathers in China due to the large amount of fowl consumed. As for the scientific reasons to study this bacteria, most of the thermophiles are in the Archaea domain, but Meiothermus ruber is a unique thermophile in the Eubacteria domain.What is a Genome Annotation?A Genome Annotation is a process of attaching biological information to DNA sequences

7An annotation is the information that you find through using the programs that JGI gives you. These programs look at the gene in many different ways such as the DNA compared to other organisms to determine if there are any resemblances between Meiothermus ruber gene DNA and another organism gene DNA. Also these programs can find out what that gene's structure, location, and uses are and much more. You will only go through the first three modules, but there are several more modules that can find out more about the gene that will be assigned to you. Once you have went through the programs with your gene, you will end up with an annotation with the information about the gene DNA that will give to the big picture of your assigned gene.

Why Annotate More Genomes?ArchaeaBacteria

8Most of what we know comes from a relatively small subset of lifes diversity. We essentially want to learn as much as we can about genomes of every kind of species out there! The more we know, the more we benefit. For example, there can be millions of genes in a genome. By annotating each of them, it can help us better understand why certain genes function the way they do. If we figure out their specific functions, we can find the specific reason why the genes are placed in an organism the way the are. This can lead to major medical success such as fighting off pathogens or even finding a cure for a lethal disease. GEBA Genomes

*T.P. Curtis, W.T. Sloan, and J.W. Scannell. 2002. Estimating prokaryotic diversity and its limits. Proc Natl Acad Sci USA 99: 10494-10499. Genomic Encyclopedia of Bacteria & Archaea (GEBA) is a massive JGI genome sequencing effort to fill in many of the missing or under-sampled branches of the Bacteria & Archaea trees.

9Many genomes have been sequenced by JGI in an effort called the Genomic Encyclopedia of Bacteria and Archaea. However, Genomes such as that of Meiothermus ruber have not been sequenced completely. Sequencing missing or undersampled branches of the Bacteria and Arachea tree are invaluable in research because it can lead to exciting findings.* D. Wu, P. Hugenholtz, K. Mavromatis, et al., 2009. A phylogeny-driven genomic encyclopedia of Bacteria and Archaea. Nature 462: 1056-1060. First 56 GEBA genomes* filled in several missing or under-sampled branches of the Bacteria trees & showed that there is a lot of genomic diversity out there to be discovered.

GEBA continued10The efforts of the JGI project, GEBA have already sequenced many genomes. The current count is 56 and it is growing. This figure depicts the genomic diversity that can be discovered in the future.MEIOTHERMUS RUBER GENOME ANNOTATION PROJECTGenome annotation - the process of attaching biological information to DNA sequencesIt consists of two main steps:identifying elements on the genome, a process called Gene Calling, and attaching biological information to these elementsTechnology is called Bioinformatics using computer programs to analyze sequence information and make predictions

Functional genomics benchtop researchGene cloning to isolate the gene of interest from the genomeMutational studies to confirm biological function predictions

11Basically, a genome annotation is the process of giving DNA sequences, amino acid sequences, etc. biological significance. So now that we know what the heck a genome annotation is (process of attaching biological information to DNA sequences), lets dig a little deeper. Genome annotation contains two main steps. The first step is to identify elements such as e-value, isoelectric point, and amino acid sequence length on the genome. This process is called Gene Calling. The second process is to attach biological information to these elements. IMG-ACT, which you will be using for your annotation, is a pretty neat toolkit created so that people like you and me, undergraduates, can annotate!The predictions found through bioinformatic programs such as IMG-ACT can be confirmed through functional genomics or benchtop research ( lab work). Labwork may include gene cloning to isolate the gene of interest from the genome of the organism. Mutational studies on the gene can also be utilized to confirm biological function predictions.

M. ruber Genome Project

Is there evidence to support the predictions related to my gene?

Large gaps in the types of bacterial genomes studied

Learn the tools to analyze your gene prediction

Use the tools to collect evidence to support/refute the prediction

Form your argument

12Any good research project will involve in some way the inquiry wheel. For example, the Meiothermus ruber genome project will involve the inquire wheel. In this project "defining the problem" is the problem of having large gaps in the types of genomes studied. A question such as," is there evidence to support the predictions related to my gene is an example of " forming the question". Research is great, but if the research has been done before and there isn't much too add onto it conducting research is futile. Therefore it is very important to " investigate the known". In the M. ruber project " investigating the known" corresponds to learning the tools to analyze your gene prediction. Confirming your prediction can only truly be done in the lab using tools ( such agarose gels, PCR) to collect evidence. If you end up working on this project, hopefully, the end result of the inquiry wheel is forming your argument based on the evidence found and communicating your findings.It is important to note that the inquiry wheel is not necessarily followed linearly. For example, if your argument is flawed and you might go back to " carrying out the study".

IMG-ACT

Phobius

NCBI

T-Coffee

BLAST

Web Logo

KEGG

PSORT

SignalP

TIRGfam

Phylogeny.fr

TMHMM

13There are many different resources out there to aid in the annotation of genes. A few of the major ones re listed on this slide. For this project you will be using the IMG-ACT database, BLAST, T-Coffee, Phobius, Web Logo, and PSORT to do the initial stages of a an annotation. These databases and search engines are just a few of the many resoources you will have to use when trying to find information on the gene you are researching.Why Annotate with Students?Most automated genome annotations - 35% are wrongAutomated annotations miss things!Learning new and valuable information is keyPrevious knowledge can help you!

14Unfortunately, around 35% of automated genome annotations are wrong and contain many mistakes. just like all other electronic devices, they are not perfect, and they can miss important things!Another important reason why students should annotate is because it will teach them invaluable information and skills that they might use later on in life. It can be difficult at times, but is extremely rewarding work. Not only do you learn new information, but previous knowledge can help you tremendously while doing this project.Annotation GoalsDevelop and strengthen genome annotation skills such as: Using computer programs to analyze sequence data Gathering and evaluating information from Web-based community-accessible sequence databasesEvaluating automated gene calls Produce quality annotations for incorporation into the Integrated Microbial Genomes Database

Build conceptual understanding of: Evolutionary relationships among genomes Genome organization Power and limitations of bioinformatics Protein structure and function Transcriptional and translational signals

Develop basic scientific research skills such as: Reading and evaluating primary literature Developing hypotheses and interpreting data Drawing conclusions from a collection of evidence Working collaboratively Working with real data

15The overall goals for this annotation project are to develop and strengthen genome annotation skills such as:.........., To produce quality annotations for incorporation into the integrated microbial genome database, to build and strengthen a conceptual understanding of............................, and to develop basic scientific research skills such as:IMG-ACT Modular AnnotationStreamline annotationEmphasizes biological root of bioinformaticsMore easily compatible with educationEmphasizes complementarity of toolsAllows addition and removal of modules to match student level

16So, by now you are probably wondering how you are actually going to perform annotations. Without outside help, annotation takes a lot of hard work, some of which may not seem relevant to your studying cell bio. However, to aid in this task, these things called "modules" have been developed to guide you through the annotation project. For instance, by using these modules, you'll be able to understand how annotation fits in with DNA structure and gene expression while carrying over those skills from module to module. Essentially, although these bioinformatics databases may seem complex at times, they are still underlied by a basic understanding of how biology works on the cellular level. With the modules developed for the annotation project the connection between what you have learned in lecture and what you are learning through annotation will become much clearer.ANNOTATIONModule TitleDescriptionMod 1: Basic InformationDNA coordinates & base sequence, amino acid sequence, pIMod 2: Sequence-based Similarity DataSequence alignment, conserved protein domains and protein familiesMod 3: Cellular Localization Data Signal peptide sequence, transmembrane domains

17Now that you are all pumped up for this annotation project, we should probably explain to you what information you are actually going to be dealing with. You are going to be assigned a specific gene from M. ruber to annotate. The first thing you have to do in your annotation is to find out the basic specs of your gene. This information mainly concerns how big your gene is, what amino-acid sequence it codes for, among other important physical properties. Then, once you have that information down, you can move on to relating your gene to other genes. You'll do this by comparing your gene's amino acid sequence to other organism's amino acid sequence to see if your gene performs similar functions in other organisms. Think of this like a family photo. Basically, your comparing your gene ie photo, to other genes that look similar. Since form follows function, a common gene form often points to a common gene function. The last thing your going to do is find where the protein coded by the gene is found. This basically consists of finding the postal address attached to your gene's proteinwhich tells it where to go.Module ConceptsBasic Information

18With all of this talk of annotation and modules, by now you're probably wondering what all of this wonderful biology research actually looks like. It actuallylooks, well, quite normal. Basically, from thelist of gene numbers,you'll pick the one that your instructor gives to you.An important thing to remember is thatwhen you are using thiswebsite, alwaysuse multiple tabs. Sometimes if you click on a link,the browser wont let you go to the previous slide, which canbe the catalyst for a major freakout. Once you have all of these tabs open, you can follow along with your modules in one screen andthe annotationproject screens in another. By doing this, you can fill out information in your lab notebook and switch to your modules really fast. Typically this project takes a few hours, so make sure to usethose handy computer shortcuts to makeyour annotation goquick and smooth.For some of the annotationyou will be redirected to an outside webpage. When that happens, just make sure to keep your lab notebook and modules openso you can refer to themon short notice. IMG-ACT (JGI):Cheryl Kerfeld (ckerfeld@lbl.gov)Seth Axen (saxen@lbl.gov)www.jgi.doe.gov/education/annotation_tools.html

Microbial Genome Annotation Network:(NSF RCN-UBE)Lori Scott, PI (LoriScott@augustana.edu)mgan.jgi-psf.org

Acknowledgements

19Lastly, there is one important thing you need to know about biological research and research in general, it often requires a lot of teamwork. This project was set up by a division of the Department of Energy for the reason of researching extremophiles. The information gleaned from the data you obtain may be useful to other researchers trying to apply genomic data to their own studies. Also, this project is not just one localized around Augustana. Other big name colleges like the University of Nebraska and MU are also doing similar projects to introduce undergraduates to the new tools available to geneticists in the digital age. So take pride in your work! To say that annotation is the funnest thing in the world would be an exagerration. But it does have relevance to what you'll be studying as a biologist for years to come.

The future is now!!!The End!

Thanks for listening to our project. We hope you guys feel as enthusiastic about as we do. This is a really exciting time in the field of genetics where lab research is taking the next step into the information age. As you continue your studies in Biology, we hope this information will continue to be relevant to your work. So get going and start your annotations!!!! 20