Genome Annotation

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Genome Annotation

• BBSI
• July 14, 2005
• Rita Shiang

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Genome Annotation

• Identification of important components in genomic
  DNA

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What is a Gene?

• Fundamental unit of heredity
• DNA involved in producing a polypeptide it
  includes regions preceding and following the
  coding region (leader and trailer) as well as
  intervening sequences (introns)
• Entire DNA sequence including exons, introns, and
  noncoding transcription-control regions

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What Components are Important in Protein Coding
Genes?

• Sequences that initiate transcription
• Sequences that process hnRNA to mRNA
• Signals important in translation

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TATA Box
Lodishet al, Molecular Cell Biology, 2000, Fig.
10.30.
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Other Promoters

• Initiator consensus
• 5Py Py A(1) N T/A Py Py Py
• N A, T, G or C
• Py pyrimidine C or T
• GC rich sequences
• Stretch of 20-50 GC nucleotides 100 bp upstream
  of start site (CpG not common in genome)
• Housekeeping genes
• Multiple initiation sites

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Polyadenylation Cleavage

• Addition of a string of As to mRNAs
• Polyadenylation signal AAUAAA found before
  cleavage site
• GU or UU rich region 50 bp from the cleavage
  site
• Stabilizes mRNA transcripts

Lodishet al, Molecular Cell Biology, 2000, Fig.
11.23.
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Splicing
Electron micrograph of adenovirus DNA and hexon
gene mRNA
Lodishet al, Molecular Cell Biology, 2000, Fig.
11,13.
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Splice Reaction
Lodishet al, Molecular Cell Biology, 2000, Fig.
11.15.
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Splice Sites
Lodishet al, Molecular Cell Biology, 2000, Fig.
11,14.
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Additional Splice Sites
Consensus Py7NCAG-G(exon)AG GUAAGU
98.12 Nonconsensus GC U12 introns
AC PuUAUCCUPy 0.76 Other rare
sequences 1 Py C or U Pu A or G
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Translation Signals

• 5 Cap structure directs ribosomal binding
• AUG codes for methionine. The first AUG in a
  transcript is where translation starts
• Open reading frame (ORF)
• Stretch of sequence that codes for amino acids
  before a stop codon
• Translation stop codons UAG, UAA, UGA

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Capping of 5RNA with 7-methylguanylate (m7G)
Lodish et al, Molecular Cell Biology, 2000, Fig.
11.8.
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Known Gene Components
Lodishet al, Molecular Cell Biology, 2000, Fig.
10.34.
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Genome Annotation

• What is in a genome besides protein coding genes?

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Repetitive DNA makes up at least 50 of the genome

• Transposon-derived interspersed repeats
• Inactive retroposed copies of genes pseudogenes
• Simple short repeats
• Segmental Duplications
• Blocks of tandemly repeated sequences
• Centromeres
• Telomeres
• Short arm of acrocentric chromosomes
• Ribosomal gene clusters

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Non-protein coding genes or non-coding RNA (ncRNA)

• tRNA genes
• rRNA genes
• snRNA genes
• Splicing
• Telomere maintenance
• snoRNA genes
• Other
• microRNA

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Annotation of Genomic DNA

• Identifying Protein Coding Genes
• Placing the genes on the genome (where are they?)

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How Many Genes in the Genome?

• Early on based on reassociation kinetics the
  estimate was 40,000
• Walter Gilbert estimated 100,000 based on gene
  and genome size
• 70,000 80,000 based on an extrapolated number
  of CpG islands
• With the Human sequence the estimate is 30,000
  40,000

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Annotation of Genomic DNA Specifically for Genes
that Code for Proteins

• Match genomic DNA to genes that have been
  previously cloned and sequenced looking for
  sequence similarity using BLAST programs
• Predict genes using computer programs to scan
  genomic DNA using known elements
• Many strategies use a combination of both methods

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cDNA Library Construction
Lodishet al, Molecular Cell Biology, 2000, Fig.
7.14
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Lodishet al, Molecular Cell Biology, 2000, Fig.
7.15
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Gene AnnotationCelera

• Constructed gene models using sequence from cDNAs
  
• Used Unigene database
• Partitions GenBank sequences (mRNAs ESTs) into
  non-redundant set using 3 UTRs
• 111,064 Unigene clusters for human

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Gene AnnotationCelera cont.

• Predicts gene boundaries by identifying
  overlapping sets of EST and protein matches
• Known full-length genes were annotated on the map
  (matched w/50 of the length gt92 identity)
• Clusters that did not match a full-length gene
  were evaluated using other references
• Conservation of genomic sequence between mouse
  human
• Similarity between human rodent transcripts
• Similarity to known proteins

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Validation

• Validated by construction of known genes (RefSeq)
• 6.1 of RefSeq genes were not annotated by Otto

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Gene Annotation - Human Genome Sequencing
Consortium

• Start with Ensemble predicted genes
• ab initio predictions using Genscan
• Based on probabilistic model of genome sequence
  composition and gene structure
• Confirm similarity to mRNAs, ESTs, protein motifs
  from all organisms
• Extend protein matches using GeneWise
• Compares protein based information to genomic
  sequence and allows for frameshifts and large
  introns
• Produces partial gene predictions

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Consortium cont.

• Merge Ensemble gene predictions w/ Genie
  predictions
• Genie identifies matches of mRNAs and ESTs
• Employs hidden Markov models (HMMs) to extend
  matches using ab initio statistical methods
• Links information from 5 and 3 ESTs from the
  same cDNA clone to complete a sequence from the
  ATG to the stop codon
• Can generate alternatively spliced products
  (though only longest used in this build)
• Merge results with genes in RefSeq, SWISSPROT and
  TrEMBL databases

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Validation

• Validate method by comparing to a new set of
  known genes, a set of mouse cDNAs and genes on
  Chromosome 22 (Finished Sequence)
• 85 Sensitivity
• 13 spurious predictions

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Factors Affecting Gene Annotation

• Splice sites do not conform to consensus
• Noncoding exons are common
• Exon what is left over after splicing after
  introns are removed and does not refer to a
  stretch of coding information
• tRNAs are spliced but noncoding
• gt35 of human genes have noncoding exons
• No statistical bias so they are difficult to
  identify

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Factors Affecting Gene Annotation Cont.

• Internal exons can be very small
• Avg. size of internal exons are 130 bp
• 65 of vertebrate exons are 68-208 bp
• gt10 are lt60 bp
• Exons lt 10 bp have been identified
• Invected gene in Drosophila
• One of four exons is 6 bp (GTCGAA)
• Flanked by introns of 27.6 and 1.1 kb
• Not correctly recognized by cDNA alignment
  software and creates a frameshift in the gene
• Exons of size 0
• Resizing exons create an intermediate splice
  product

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Places to View Annotated Genomes

• National Center for Biotechnology Information
  (NCBI)
• Ensemble
• The Golden Path (UCSC Genome Browser)
• Celera

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Verification of Annotation in C. elegans by
Experimentation

• Complete genomic sequence
• Small introns
• Small intergenic regions

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(No Transcript)
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Results

• 11,984 cDNAs successfully cloned out of a
  prediction of 19,477
• 4,365 were not represented by cDNAs or ESTs
• Failure of cloning could be due to
• Wrongly predicted exons
• Very low expressing genes
• Not a real gene

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Verification of intron/exon structures
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Comparison of a Single Transcript
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Greater than 50 of intron/exon structures need
correcting?