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Functional Annotation & Comparative Genomics Lavanya Rishishwar February 26 th, 2014 26th Feb 20141.

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Presentation on theme: "Functional Annotation & Comparative Genomics Lavanya Rishishwar February 26 th, 2014 26th Feb 20141."— Presentation transcript:

1 Functional Annotation & Comparative Genomics Lavanya Rishishwar February 26 th, th Feb 20141

2 Outline Functional annotation What is functional annotation? Importance of functional annotation Approaches to functional annotation Pros/cons of available approaches Comparative genomics What is comparative genomics? Importance of comparative genomics Approaches and tools 26th Feb

3 THE ‘WHAT?’ Functional Annotation 326th Feb 2014

4 Genome Assembly Assemble the Pieces Right 426th Feb 2014

5 Gene Prediction W hen on board HMS Beagle, as naturalist, I was much struck with certain facts in the distribution of the inhabitants of South America, and in the geological relations of the present to the past inhabitants of that continent. These facts seemed to me to throw some light on the origin of species - that mystery of mysteries, as it has been called by one of our greatest philosophers. Identify the words W hen on board HMS Beagle, as naturalist, I was much struck with certain facts in the distribution of the inhabitants of South America, and in the geological relations of the present to the past inhabitants of that continent. These facts seemed to me to throw some light on the origin of species - that mystery of mysteries, as it has been called by one of our greatest philosophers. 526th Feb 2014

6 Functional Annotation W hen on board HMS Beagle, as naturalist, I was much struck with certain facts in the distribution of the inhabitants of South America, and in the geological relations of the present to the past inhabitants of that continent. These facts seemed to me to throw some light on the origin of species - that mystery of mysteries, as it has been called by one of our greatest philosophers. nat·u·ral·ist [nach-er-uh-list, nach-ruh-] noun 1. a person who studies or is an expert in natural history, especially a zoologist or botanist. 2. an adherent of naturalism in literature or art. Origin: 1580–90; natural + -ist Origin of Species, The noun ( On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life ) a treatise (1859) by Charles Darwin setting forth his theory of evolution. Identify the function (i.e., meaning) of each word DATABASES PROFILES 626th Feb 2014

7 Comparative Genomics W hen on board HMS Beagle, as naturalist, I was much struck with certain facts in the distribution of the inhabitants of South America, and in the geological relations of the present to the past inhabitants of that continent. These facts seemed to me to throw some light on the origin of species - that mystery of mysteries, as it has been called by one of our greatest philosophers. 726th Feb 2014 W hen on board RMS Titanic, as painter, I was much struck with certain facts in the distribution of the inhabitants of United Kingdom, and in the socioeconomical relations of the present to the past inhabitants of that continent. These facts seemed to me to throw some light on the origin of capitalism- that mystery of mysteries, as it has been called by one of our greatest philosophers.

8 THE GRAVITY OF THE ANNOTATION PROCESS Not just Newtonian 826th Feb 2014

9 “Ultimately, one wishes to determine how genes—and the proteins they encode—function in the intact organism.” Albert B, et al. (2002) Molecular biology of cell. New York: Garland Science. function 926th Feb 2014

10 Function? What is it? To a cell biologist function might refer to the network of interactions in which the protein participates or to the location to a certain cellular compartment. To a biochemist, function refers to the metabolic process in which a protein is involved or to the reaction catalyzed by an enzyme. 1026th Feb 2014

11 Functional Annotation Functional annotation consists of attaching biological information to genomic elements. Biochemical function Biological function Involved regulation and interactions Expression 1126th Feb 2014

12 What needs to be annotated? Proteins – – Domain/Motifs – Signaling Peptide – Transmembrane region Coding and non-coding RNAs Operons 26th Feb

13 Domain/Motif Domain: A discrete structural unit that is assumed to fold independently of the rest of the protein and to have its own function. ~ aa Motif: Are short, conserved regions and frequently are the most conserved regions of domains. Motifs are critical for the domain to function. 26th Feb

14 Coding and non coding RNA’s Protein Coding Enzymes Structural Regulatory Signal Transduction Receptors Toxins Virulence Factors Membrane/ Transmembrane Non Coding Riboswitches CRISPR Srna's Pathway Prediction 26th Feb

15 How Gene Performs Function? Operon Operon: Several genes with related functions that are regulated together, because one piece of mRNA codes for several related proteins. Polycistronic mRNA, mRNA coding for more than one polypeptide, is found only in prokaryotes 26th Feb

16 APPROACHES TO FUNCTIONAL ANNOTATION An overview 1626th Feb 2014

17 Approaches to functional annotation Ab initio Based on intrinsic characteristics of gene/protein features – Signaling peptides (SignalP, LipoP) – Transmembrane domains (TMHMM) Homology Based Information transfer from experimentally characterized system – BLAST – InterPro 26th Feb

18 Ab initio approaches Fairly standard – TM and Signaling peptides have a distinct pattern of sequence composition TM proteins are membrane bound receptors and channels that are of particular pharmacological relevance (therapeutic or vaccine target) Signal peptides direct proteins to their proper cellular or extracellular location 26th Feb

19 Homology based approaches Significant sequence similarity implies homology or shared ancestry that often leads to shared function Assumption – – Genes/proteins evolved to perform some function will retain that function – Deleterious mutations will be weeded out by purifying selection – Evolution is mostly dominated by divergence – Homology will thus entail a high chance of shared origin and function 26th Feb

20 Homology based approaches Databases: – NCBI GenBank RefSeq – EBI SwissProt UniProt – DDBJ KEGG Tools – BLAST – InterProScan – GO-based 26th Feb

21 Databases 26th Feb

22 Primary vs derivative sequence databases 26th Feb Sequence DataGenBank From Sequencing Labs RefSeq Genomes UniGene Curators Assemblies Computational Algorithms PGAAP

23 Database choices RefSeq, SwissProt and UniProt are all – Very reliable – High level of annotation – Minimal redundancy – Integration with other databases 26th Feb

24 Gene Ontology 26th Feb Shulaev, V., Sargent, D. J., Crowhurst, R. N., Mockler, T. C., Folkerts, O., Delcher, A. L.,... & Salama, D. Y. (2010). The genome of woodland strawberry (Fragaria vesca). Nature genetics, 43(2),

25 Analysis Tools - BLAST 26th Feb God help you if you do this here.

26 Analysis Tools - InterProScan 26th Feb

27 Analysis Tools - InterProScan 26th Feb

28 Analysis Tools – GO Based Blast2GO GOMiner …? 26th Feb

29 Criteria for selecting methods 1.Currently being maintained 2.Applicable to Prokaryotic sequences 3.Could be installed locally (support batch jobs if GUI) OR Could be included in a pipeline i.e., have a command-line interface 2926th Feb 2014

30 Gene naming You need to have a clear logic and support for assigning names to the predicted proteins A generally accepted scheme is as follows: – High confidence matches – function and annotation can be transferred – Multiple high confidence matches – assign a less specific name e.g. ABC transporter – Low confidence matches – assign function as putative – Match to a hypothetical protein – conserved hypothetical protein – No match in the database – hypothetical protein How high is high? Depends on your data. 26th Feb

31 Automated Pipelines Takes in whole genome assembly and spits out annotations. E.g.: PGAAP – Prokaryotic Genome Automatic Annotation Pipeline CG-Pipeline – Computational Genomics Pipeline RAST – Rapid Annotation using subsystem technology KEGG – Kyoto Encyclopedia of Genes and Genomes And more comes out each year with specific focus and capability 26th Feb

32 CAUTION! PROS AND CONS OF CONVENTIONAL APPROACHES Choosing The Right Function Prediction Tool 3226th Feb 2014

33 33

34 “Perutz et al. showed in 1960 that myoglobin and hemoglobin, the first two protein structures to be solved at atomic resolution using X-ray crystallography, have similar structures even though their sequences differ.” 26th Feb

35 Pros and Cons: There are no free lunches! Homology Useful but different from “same” function – Simply implies common ancestry Punta, M., & Ofran, Y. (2008). The rough guide to in silico function prediction, or how to use sequence and structure information to predict protein function. PLoS computational biology, 4(10), e

36 Pros and Cons: There are no free lunches! 3626th Feb 2014 Punta, M., & Ofran, Y. (2008). The rough guide to in silico function prediction, or how to use sequence and structure information to predict protein function. PLoS computational biology, 4(10), e

37 Pros and Cons: There are no free lunches! Again: Quality of prediction is as good as the quality of annotation of the database Eukaryotic function predictor can not be used for Prokaryotes and vice versa Building pan-genomes is a good strategy for finding more confident matches 3726th Feb 2014

38 COMPARATIVE GENOMICS 26th Feb

39 Comparative Genomics 26th Feb Ciccarelli, F. D., Doerks, T., Von Mering, C., Creevey, C. J., Snel, B., & Bork, P. (2006). Toward automatic reconstruction of a highly resolved tree of life.Science, 311(5765),

40 Comparative Genomics In a nutshell – it’s comparing similarities and differences in genomes (proteins/genes/SNPs) of multiple organisms from same or different species. Helps in answering – – Present: lifestyle - virulent vs avirulent; horizontally acquired segments – Past: Evolution 26th Feb

41 Comparative Genomics Biological questions of general interest: – Are there are rearrangements? – Is the region(s) of interest syntenic across species? – Are their gene gain/loss event leading to specific trait? – What organisms are more similar? What are most distant? – What factors confer virulence to the genome? – In our case: capsule switching? What, why and how? 26th Feb

42 Comparative Genomics Darling, Aaron E., István Miklós, and Mark A. Ragan. "Dynamics of genome rearrangement in bacterial populations." PLoS Genetics 4.7 (2008): e Genomic Rearrangement 26th Feb

43 Comparative Genomics Krause, A., Ramakumar, A., Bartels, D., Battistoni, F., Bekel, T., Boch, J.,... & Goesmann, A. (2006). Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72. Nature biotechnology, 24(11). Synteny 26th Feb

44 Comparative Genomics Horizontal Gene Transfer 26th Feb

45 Comparative Genomics You are going to hear more about your specific goals next week Remember: The focus here is not about the tools but (1) identification of the biological question, (2) your approach to answering the question and (3) your results with interpretation 26th Feb

46 Databases As before – there are number of sequence databases available – You need to decide what subset of that database should you taking into consideration – For e.g.: what organism/serogroup/sequence type should your database be focused on? If we are also looking for virulence factors - VFDB If we are interested in pathways – KEGG, Pathway Tools 26th Feb

47 Analysis Tools Homology Based – BLAST, Protein Clusters, Pathway Analysis Phylogenetics – MEGA, T-Coffee Virulence - VFDB Horizontal/Lateral Gene Transfer – Dark Horse, Alien Hunter, Phylogeny Based Visualization 26th Feb

48 Phylogenetic Analysis There are a number of ways you can compare organisms/genomes: – 16S rRNA tree – MLST based methods – ANI based methods All three can be visualized as a tree to assess the relatedness between the organisms ANI has been shown to correlate well with DDH by Konstantinidis et al More traditional Konstantinidis, K. T., Ramette, A., & Tiedje, J. M. (2006). The bacterial species definition in the genomic era. Philosophical Transactions of the Royal Society B: Biological Sciences, 361(1475), Goris, J., Konstantinidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P., & Tiedje, J. M. (2007). DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. International journal of systematic and evolutionary microbiology, 57(1), th Feb

49 Different phenotype, same evolutionary lineages Phenotypic concordance need not support same ancestral lineage At times it has been observed that species tend to gain certain set of mutations in same or different gene(s) which leads to the same phenotype Acquiring antibiotic resistance is one such example The investigation of such cases depends on a case-by-case manner with underlying reasons varying from SNPs, gene gain/loss, indels, plasmid uptake etc 26th Feb

50 Visualization It’s important than you think Plethora of visualization tools are available today for various purposes E.g.: – Circos – CGView – BRIG – Artemis – IGV – Mauve – VISTA, etc 26th Feb

51 Visualization 26th Feb

52 Visualization 26th Feb

53 Visualization 26th Feb Rishishwar, L., Katz, L. S., Sharma, N. V., Rowe, L., Frace, M., Thomas, J. D.,... & Jordan, I. K. (2012). Genomic Basis of a Polyagglutinating Isolate of Neisseria meningitidis. Journal of bacteriology, 194(20),

54 Capsule switching breakpoint resolution 26th Feb Rishishwar, L., Katz, L. S., Sharma, N. V., Rowe, L., Frace, M., Thomas, J. D.,... & Jordan, I. K. (2012). Genomic Basis of a Polyagglutinating Isolate of Neisseria meningitidis. Journal of bacteriology, 194(20),

55 26th Feb


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