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Genome Annotation. Now that you’ve assembled your genome, what is next? GENOME ANNOTATION What is that? Why is it important? How do you do it?. Challenges to Genome Annotation ?. Finding genes involves computational methods as well as experimental validation

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Challenges to genome annotation
Challenges to Genome Annotation?

  • Finding genes involves computational methods as well as experimental validation

  • Computational methods are often inadequate, and often generate erroneous ‘gene’ (false positive) sequences which:

    • Are missing exons

    • Have incorrect exons

    • Over predict genes

    • Where the 5’ and 3’ UTR are missing


What kinds of things are we looking to annotate
What kinds of things are we looking to annotate?

  • CDS - coding sequences

  • mRNA

  • Alternative RNA

  • Promoter and Poly-A Signal

  • Pseudogenes

  • ncRNA


Pseudogenes
Pseudogenes

  • Could be as high as 20-30% of all Genomic sequence predictions could be pseudogene

  • Non-functional copy of a gene

    • Processed pseudogene

      • Retro-transposon derived

      • No 5’ promoters

      • No introns

      • Often includes polyA tail

    • Non-processed pseudogene

      • Gene duplication derived

    • Both include events that make the gene non-funtional

      • Frameshift

      • Stop codons

  • We assume pseudogenes have no function, but we really don’t know!


Noncoding rna ncrna
Noncoding RNA (ncRNA)

  • ncRNA represent 98% of all transcripts in a mammalian cell

  • ncRNA have not been taken into account in gene counts

    • cDNA

    • ORF computational prediction

    • Comparative genomics looking at ORF

  • ncRNA can be:

    • Structural

    • Catalytic

    • Regulatory

  • tRNA – transfer RNA: involved in translation

  • rRNA – ribosomal RNA: structural component of ribosome, where translation takes place

  • snoRNA – small nucleolar RNA: functional/catalytic in RNA maturation

  • Antisense RNA: gene regulation/silencing?


  • Covariance model searches are extremely compute intensive. A small model (like tRNA) can search a sequence database at a rate of around 300 bases/sec. The compute time scales roughly to the 4th power of the length of the RNA, so larger models quickly become infeasible without significant compute resources.


    Blast
    BLAST small model (like

    • Seeks high-scoring segment pairs (HSP)

      • pair of sequences that can be aligned without gaps

      • when aligned, have maximal aggregate score (score cannot be improved by extension or trimming)

      • score must be above score threshold S

    • Public Search engines

      • WWW search formhttp://www.ncbi.nlm.nih.gov/BLAST

      • Unix command lineblastall -pprogname -d db -i query > outfile


    Which matrix
    Which Matrix? small model (like

    • Triple-PAM strategy (Altschul, 1991)

      • PAM 40 Short alignments, highly similar

        • tblastn against ESTs

      • PAM 120

      • PAM 250 Longer, weaker local alignments

        • Looking in the twilight zone

    • BLOSUM (Henikoff, 1993)

      • BLOSUM 90 Short alignments, highly similar

      • BLOSUM 62 Most effective in detecting known members of a protein family

        • Standard on NCBI server – works in most cases

      • BLOSUM 30 Longer, weaker local alignments


    Protein coding genes in prokaryotes and simple eukaryotes
    Protein coding genes in prokaryotes, and simple eukaryotes small model (like

    • Use ORF finder

      http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi

    • Simple ATG/Stop

    • Simple link to FASTA formatted files and BLAST.

    • Problems:

      • In frame Methionine

      • Small protein

    • Solution: comparative genomics


    Figure 11 from: Methods in comparative genomics: genome correspondence, gene identification and regulatory motif discovery. Kellis M, Patterson N, Birren B, Berger B, Lander ES. J Comput Biol. 2004;11(2-3):319-55.

    Saccharomyces cerevisiae.

    Saccharomyces paradoxus,

    Saccharomyces mikatae,

    Saccharomyces bayanus


    Ab initio gene identification
    Ab correspondence, gene identification and regulatory motif discovery. initio gene identification

    • Goals

      • Identify coding exons

      • Seek gene structure information

      • Get a protein sequence for further analysis

    • Relevance

      • Characterization of anonymous DNA genomic sequences

      • Works on all DNA sequences


    Gene finding strategies
    Gene-Finding Strategies correspondence, gene identification and regulatory motif discovery.

    Genomic Sequence

    Content-Based

    Site-Based

    Comparative

    • Inferences basedon sequence homology:

    • Protein sequence with similarity to translated product of query

    • Modular structure of proteins usually precludes finding complete gene

    • Bulk properties ofsequence:

    • Open reading frames

    • Codon usage

    • Repeat periodicity

    • Compositional complexity

    • Absolute properties ofsequence:

    • Consensus sequences

    • Donor and acceptor splice sites

    • Transcription factor binding sites

    • Polyadenylation signals

    • “Right” ATG start

    • Stop codons out-of-context


    Gene finding methods
    Gene-Finding Methods correspondence, gene identification and regulatory motif discovery.

    Genomic Sequence

    Rule-Based

    Neural Network

    • Cutoff method:

    • Criteria applied sequentially to identify possible exons

    • Rank or eliminate candidates from consideration based on pre-determined cutoff at each step

    • Composite method:

    • Criteria applied in parallel

    • Training sets used to optimize performance

    • Weight scores in order of importance


    Evaluation statistics
    Evaluation Statistics correspondence, gene identification and regulatory motif discovery.

    TP

    TP

    TN

    FN

    FP

    TN

    FN

    Actual

    Predicted

    Sensitivity Fraction of actual coding regions that are correctly predicted as coding

    Specificity Fraction of the prediction that is actually correct

    Correlation Combined measure of sensitivity and specificity,

    Coefficient ranging from –1 (always wrong) to +1 (always right)


    The process
    The Process correspondence, gene identification and regulatory motif discovery.

    • Compute the prediction

    • Confirm with biological sequences (also with computational tools)

    • Integrate all of this

    • Annotate genome (often via a GUI: Graphical User Interface)

    • Validate

    • Re-annotate/Update

    • Check it twice

    • Submit to GenBank


    Lots of software
    Lots of Software: correspondence, gene identification and regulatory motif discovery.

    • EnsEMBL (EBI)

    • Sequin (NCBI)

    • PseudoCAP (SFU)

    • GMOD (CSHL)

    • Pegasys (UBiC)

    • Apollo (EBI/Berkeley)

    • GeneMark (Georgia Institute of Tech)

    • GeneScan (MIT)

    • GenomeThreader (University of Hamberg)

    • HMMgene (Technical University of Denmark)


    Genbank features
    GenBank correspondence, gene identification and regulatory motif discovery. Features

    -10_signal

    -35_signal

    3'clip

    3'UTR

    5'clip

    5'UTR

    attenuator

    CAAT_signal

    CDS

    conflict

    C_region

    D-loop

    D_segment

    enhancer

    exon

    GC_signal

    gene

    iDNA

    intron

    J_segment

    LTR

    mat_peptide

    misc_binding

    misc_difference

    misc_feature

    misc_recomb

    misc_RNA

    misc_signal

    misc_structure

    modified_base

    mRNA

    N_region

    old_sequence

    polyA_signal

    polyA_site

    precursor_RNA

    primer_bind

    prim_transcript

    promoter

    protein_bind

    RBS

    repeat_region

    repeat_unit

    rep_origin

    rRNA

    satellite

    scRNA

    sig_peptide

    snoRNA

    snRNA

    S_region

    stem_loop

    STS

    TATA_signal

    terminator

    transit_peptide

    tRNA

    unsure

    variation

    V_region

    V_segment


    Genbank features the important ones
    GenBank correspondence, gene identification and regulatory motif discovery. Features: the important ones

    -10_signal

    -35_signal

    3'clip

    3'UTR

    5'clip

    5'UTR

    attenuator

    CAAT_signal

    CDS

    conflict

    C_region

    D-loop

    D_segment

    enhancer

    exon

    GC_signal

    gene

    iDNA

    intron

    J_segment

    LTR

    mat_peptide

    misc_binding

    misc_difference

    misc_feature

    misc_recomb

    misc_RNA

    misc_signal

    misc_structure

    modified_base

    mRNA

    N_region

    old_sequence

    polyA_signal

    polyA_site

    precursor_RNA

    primer_bind

    prim_transcript

    promoter

    protein_bind

    RBS

    repeat_region

    repeat_unit

    rep_origin

    rRNA

    satellite

    scRNA

    sig_peptide

    snoRNA

    snRNA

    S_region

    stem_loop

    STS

    TATA_signal

    terminator

    transit_peptide

    tRNA

    unsure

    variation

    V_region

    V_segment


    Gene prediction caveats
    Gene Prediction Caveats correspondence, gene identification and regulatory motif discovery.

    • Predictions are of protein coding regions

      • Do not detect non-coding areas (5’ and 3’ UTR)

      • Non-coding RNA genes are missed

    • Predictions are for “typical” genes

      • Must predict a beginning and an end

      • Partial or multiple genes are often missed

      • Training sets may be biased

      • Methods are sensitive to G+C content

      • Weighting of factors may be inordinately biased


    Genome annotation problems
    Genome annotation problems correspondence, gene identification and regulatory motif discovery. :

    • Assembling the genome

    • Analysis & interpretation

    • Lack of consistency from gene to gene

    • Lack of consistency from person to person

    • Lack of controlled vocabulary

    • Parts we don’t know

    • Bacteria vs mammals

    • Graphical user interface

    • Gene expression/molecular interactions

    • Dimensions

    • Updates and maintenance


    The ideal annotation of mygene
    The ideal annotation of “MyGene” correspondence, gene identification and regulatory motif discovery.

    All clones

    All SNPs

    Promoter(s)

    MyGene

    All mRNAs

    All proteins

    • All protein modifications

    • Ontologies

    • Interactions (complexes, pathways, networks)

    • Expression (where and when, and how much)

    • Evolutionary relationships

    All structures


    Some concluding remarks
    Some Concluding remarks correspondence, gene identification and regulatory motif discovery.

    • Trust but verify

    • Beware of gene prediction tools!

    • Always use more than one gene prediction tool and more than one genome when possible.

    • Active area of bioinformatics research, so be mindful of the new literature in this .


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