Tools for comparative genomics and expert annotations

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Tools for comparative genomics and expert
annotations

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Goals of this Presentation

• Introduce microbiologists to the power of NMPDR
  and SEED
• Enable users to interact with data
• Invite experts to participate in construction of
  subsystems
• Capture expert annotations via the annotation
  clearinghouse

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What is NMPDR?

• Beautified, read-only version of the SEED
• What is the SEED?
• Editable environment for assignment of function
  in the context of systems biology
• Intended to clean up legacy of errors created by
  similarity-based, automated assignment of
  function
• Manual assignment of function based on integrated
  evidence sequence similarity, functional
  clusters, phylogenetic and metabolic profiles
• Developed for the project to annotate 1000 genomes

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When Will We Have 1000 Complete Genomes?

• Depends on what is meant by complete
• Many sequencing projects will stop without
  finishing or closing the genome in one
  contiguous sequence for each replicon
• A genome is essentially complete when
• 95 - 99 of genome accurately sequenced
• 10X coverage by 454 method 5X coverage by Sanger
  method
• Assembly places 70 data in contigs at least 20
  kbp

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Bacterial Genome Facts

• First two complete genomes in 1995 were bacterial
  pathogens
• 2913 genomes started as of Sept., 2007
• 63 of total are bacteria 50 of bacteria are
  pathogens
• 4434 genomes started as of January, 2009
• 51 bacteria
• Value depends on accuracy of annotation

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Complete Genome Projects
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What is an Annotation?

• Identification of nucleotide string that could
  potentially encode a protein
• Open reading frames (ORFs) computed from stop and
  start codons, codon bias, promoters and RBS
• Assignment of a name to that gene
• Usually that of known protein with most similar
  sequence, computed from translated BLAST
• Prediction of functional role for that gene
• Function of most similar protein not always
  established with experimental evidence
• Most similar protein may not have known function
• Most similar ORF may or may not be expressed

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Problems with Standard Annotations

• 42 of H. influenzae ORFs assigned no function in
  1995
• about half of those had no sequence match in
  GenBank
• the rest matched hypothetical proteins in E.
  coli
• 58 of H. influenzae ORFs assigned function of a
  significantly similar sequence
• What was in GenBank to compare with in 1995?
• 7 of all GenBank entries were bacterial, 16 of
  those, E. coli
• many conserved hypotheticals added to database
• Paralogous members of protein families may not be
  properly discriminated
• Significantly similar enzymes may act on
  different substrates
• Assignments are transitive, many times removed
  from experimental data

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Subsystems Annotationsvs.Pipelines or Protein
Families

• What is subsystems annotation?
• humans integrating evidence within a comparative
  framework
• Whats wrong with genome-at-a-time pipelines?
• automated assignment of archived annotations to
  new genomes
• propagates uninformative and incorrect
  annotations
• Whats wrong with annotation based on protein
  families?
• emphasizes structural and phylogenetic evidence
• ignores metabolic and chromosomal contexts
• leads to ambiguity for members of large families,
  e.g. transporters

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What is a Subsystem?

• Subsystem is a generalization of pathway
• Collection of functional roles jointly involved
  in a biological process or complex
• metabolic, signaling, regulatory, structural
• Functional role is the abstract biological
  function of a gene product
• Atomic or fundamental examples
• 6-phosphofructokinase (EC 2.7.1.11)
• LSU ribosomal protein L31p
• cell division protein FtsZ
• Inclusion of gene in subsystem is only by
  functional role
• Controlled vocabulary

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Expert-Defined Subsystems

• Curator is researcher with first-hand knowledge
  of biological system
• Functional roles defined and grouped into
  subsystem and subsets by curator
• universal groups of roles include all organisms
• functional variants are subsets of roles found in
  a limited number of organisms
• often represent alternative paths or
  nonorthologous replacement
• Semi-automated assignment of function based on
  manual groundwork, sequence homology, and
  functional clustering

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Subsystem Primer

• Describe your subsystem in 150 words or lesswhy
  should these functions be considered together?
• define the emergent properties of the system
• Provide or link to a diagram that illustrates
  this subsystem
• define the graph or network
• List the reactions or relationships between these
  functional roles
• define the edges
• List the exact names and abbreviations of these
  functional roles
• define the nodes
• List the id numbers (GenBank, SwissProtany
  identifying alias) of genes that play these roles
  in one or more exemplar genomes
• examples of nodes
• Provide one or more references that support the
  assignment of function for the exemplar genes
• provide evidence

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Populated Subsystems

• Two-dimensional integration of functional roles
  with genomes
• Spreadsheet
• Columns of functional roles
• Rows of organisms
• Cells of annotated genes
• Table of functional roles with GO terms
• Diagram
• Curator notes and citations

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Simple ExampleHistidine Degradation Subsystem

• Conversion of histidine to glutamate is
  organizing principle
• Functional roles defined in table

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Subsystem Diagram

• Three functional variants
• Universal subset has three roles, followed by
  three alternative paths from IV to VI

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Subsystem Spreadsheet

• Column headers taken from table of functional
  roles
• Rows are selected genomes, or organisms
• Cells are populated with specific, annotated
  genes
• Shared background color indicates proximity of
  genes
• Functional variants defined by the annotated
  roles
• Variant code -1 indicates subsystem is not
  functional

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Missing Genes Noticed by Subsystems Annotation

• No genes were annotated ForI (EC 3.5.3.13)
  Formiminoglutamic iminohydrolase when the
  Histidine Degradation subsystem was populated
• Organisms missing ForI convert His to Glu
• Candidate genes that could perform the role
  ForI must be identified
• Strategy for finding genes is based on
  chromosomal clustering and occurrence profiling

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Finding Genes that Cluster with NfoD

• Red gene in graphic and table is NfoD of
  Xanthomonas
• Genes pictured in gray boxes located nearby NfoD
  in four or more species
• Advanced controls expands display of homologous
  regions in other genomes
• Functional Coupling score links to table of
  homologous pairs in other genomes
• Cluster button finds biggest clusters in other
  species when not clustered in subject genome

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What are Pinned Regions?

• Focus gene is number 1, colored red
• Most frequently co-localized homolog numbered 2,
  colored green
• Sets of homologous genes presented in the same
  color with the same numerical label BLASTP
  cut-off e-val 1e-20
• Numerical labels correspond to rank-ordered
  frequency of co-localization with the focus gene
• Number of regions, size of region, and cut-off
  can be re-set by user

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Candidate ForI in Context with NfoD

• Compare Regions around NfoD, red, center
• HutC, the regulator, is green, 2
• HutH, the first functional role in the subsystem,
  is blue, 4
• Candidate ForI is teal, 6, originally annotated
  as conserved hypothetical

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Annotation of ForI EC 3.5.3.13

• Metabolic context proves need for role
• Organisms missing annotated ForI degrade His to
  Glu
• Chromosomal context points to candidate
• Clusters with NfoD and other genes in subsystem
• Occurrence context supports candidate
• Organisms containing NfoD lack GluF and HutG,
  required for functional variants 1 and 2,
  respectively
• Organisms containing candidate ForI also contain
  NfoD, indicating functional variant 3
• Phylogenetic trees of candidate ForI genes are
  coherent

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Subsystems Allow Bioinformatics to Inform Bench
Research

• Subsystems point to missing or alternative genes
• Bioinformatic predictions need to be tested at
  the bench
• ForI candidate now verified experimentally
• Connections forged between bench and
  bioinformatics

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How is NMPDR distinct from NCBI?

• Corrected, functional annotations, manually
  curated in context of systems biology
• Multiple starting points for accessing data
• gene or protein name, subsystem, organism
• Search results downloadable as names or sequences
• Interactive tools for comparative analysis
• Compare regionsadjust size of region, number of
  genomes
• Subsystemsbrowse phylogenetic distribution of
  biological system color spreadsheet and diagram
• Functional clustersfind genes with conserved
  proximity
• BLASTP Hitsselect and align interesting
  sequences
• Signature genesfind genes in common or that
  distinguish user-selected groups of genomes
  groups may contain one or many

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Exploration of physical, genomic context

• Compare Regions graphic
• Focus protein highlighted red
• Color-matched orthlogs allow comparative analysis
  of functional clustering and chromosomal
  rearrangements
• Redraw the display with different number of
  genomes or different size region
• Compare Regions table
• Table is sortable and filterable with active
  column headings
• Genes with conserved proximity shown with
  functional coupling scores, fc-sc
• fc-sc (functional coupling score)
• Measures conservation of gene proximity and
  phylogenetic distance
• Link returns table listing pairs of proximal
  orthologs
• CL (find best clusters)
• Finds clusters containing the focus protein in
  other genomes
• Useful for genes without functional coupling
  scores, fc-sc

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Exploration of functional, biological context

• Populated Subsystem Spreadsheet
• Columns represent functional roles, mouse over
  header for definition
• Genomes (rows) shown may be filtered and sorted
  by name or taxonomic group
• Cells populated with specific, annotated genes
  linked to context pages
• Functional variants defined by the annotated
  roles
• Variant codes defined in notes tab
• Diagram of subsystem often provided
• Protein families
• FIGfams taken from single column of functional
  roles
• Links to structures, orthologs, literature

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NMPDR Services

• Essential Genes on Genomic Scale
• Experimentally verified in genome-wide scans of
  10 important model organisms
• Drug targets pipline to in silico screening
• essential in at least one of the NMPDR pathogens
• included in subsystems by our curators
• orthologs in the Protein Data Bank
• orthologs in a substantial number of bacterial
  priority pathogens
• Targets search flexible search forms for
  discovering novel targets based on computed
  attributes
• physical characteristics such as MW, pI
• subcellular location
• transmembrane regions and signal peptides
• subsystem, pathway, reaction
• structural motifs, protein families

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Related NMPDR Services

• RAST Genome annotation server
• Automated annotation of essentially complete
  genome sequences in a small set of long sequence
  contigs
• View results in comparative context with other
  genomes
• MG-RAST Metagenome annotation server
• Automated annotation of a very large set of very
  short DNA sequences
• View results in comparative context with other
  data sets
• Annotation Clearinghouse
• Tool to credit experts with annotation of
  specific genes and to share annotations with
  other databases
• Input is a two-column table of gene IDs and
  annotations vouched for by expert

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Who is NMPDR?

• Fellowship for Interpretation of Genomes (FIG)
• Ross Overbeek, Veronika Vonstein, Gordon Pusch,
  Bruce Parrello, Rob Edwards, Andrei Osterman,
  Michael Fonstein, Svetlana Gerdes, Olga Zagnitko,
  Olga Vassieva, Yakov Kogan, Irina Goltsman
• Argonne National Laboratory
• Rick Stevens, Terry Disz, Robert Olson, Folker
  Meyer, Elizabeth Glass, Chris Henry, Jared
  Wilkening
• Computation Institute at University of Chicago
• Daniela Bartels, Michael Kubal, William Mihalo,
  Tobias Paczian, Andreas Wilke, Alex Rodriguez,
  Mark D'Souza, Rami Aziz
• University of Illinois at Urbana Hope College
• Gary J. Olsen, Claudia Reich, Leslie McNeil
  Aaron Best, Matt DeJongh
• National Institute of Allergy and Infectious
  Diseases
• National Institutes of Health, Department of
  Health and Human Services, Contract
  HHSN266200400042C.