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    Annotating Genomes Using Proteomics Data: Andy Jones Department of Preclinical Veterinary Science

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    Shaher Bano Mirza
    This document summarizes a study on using proteomics data to improve genome annotation. It discusses how current genome annotation methods are imperfect and miss many genes and splicing events. The study analyzed the Toxoplasma gondii genome and proteome and found evidence to confirm over 1/3 of predicted genes and 2500 splicing events. However, it also found evidence supporting alternative gene models not predicted by current methods. The document proposes an automated pipeline to iteratively re-query proteomics data when gene models change, integrate peptide evidence into gene finding, and attempt de novo sequencing to further improve genome annotation.

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    Annotating Genomes Using Proteomics Data: Andy Jones Department of Preclinical Veterinary Science

    Uploaded by

    Shaher Bano Mirza
    23 views22 pages
    This document summarizes a study on using proteomics data to improve genome annotation. It discusses how current genome annotation methods are imperfect and miss many genes and splicing events. The study analyzed the Toxoplasma gondii genome and proteome and found evidence to confirm over 1/3 of predicted genes and 2500 splicing events. However, it also found evidence supporting alternative gene models not predicted by current methods. The document proposes an automated pipeline to iteratively re-query proteomics data when gene models change, integrate peptide evidence into gene finding, and attempt de novo sequencing to further improve genome annotation.

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    Proteogenomics10Oct2008 v2 com

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    This document summarizes a study on using proteomics data to improve genome annotation. It discusses how current genome annotation methods are imperfect and miss many genes and splicing events. The study analyzed the Toxoplasma gondii genome and proteome and found evidence to confirm over 1/3 of predicted genes and 2500 splicing events. However, it also found evidence supporting alternative gene models not predicted by current methods. The document proposes an automated pipeline to iteratively re-query proteomics data when gene models change, integrate peptide evidence into gene finding, and attempt de novo sequencing to further improve genome annotation.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    23 views22 pages

    Annotating Genomes Using Proteomics Data: Andy Jones Department of Preclinical Veterinary Science

    Uploaded by

    Shaher Bano Mirza
    This document summarizes a study on using proteomics data to improve genome annotation. It discusses how current genome annotation methods are imperfect and miss many genes and splicing events. The study analyzed the Toxoplasma gondii genome and proteome and found evidence to confirm over 1/3 of predicted genes and 2500 splicing events. However, it also found evidence supporting alternative gene models not predicted by current methods. The document proposes an automated pipeline to iteratively re-query proteomics data when gene models change, integrate peptide evidence into gene finding, and attempt de novo sequencing to further improve genome annotation.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 22

    Annotating genomes using

    proteomics data
    Andy Jones

    Department of Preclinical Veterinary


    Science
    Overview
    • Genome annotation
    – Current informatics methods
    – Experimental data
    – How good are we at annotating genomes?
    • Proteome data for genome annotation
    – Study on Toxoplasma
    – Challenges
    – Proposed solutions
    Summary: 780 “completed” genomes; 734 “draft” assembly; 842 “in progress”
    Total: 2356 (1996 prokaryote, 360 eukaryote)

    Genome sequencing is just a starting point to understanding genes / proteins


    Annotating eukaryotic genomes
    Start codon Stop codon
    Exon 1 Exon 2 Exon 3 Exon 4

    Genomic DNA

    mRNA

    • Genome annotation:
    – Find start codons / transcriptional initiation
    – Recognise splice acceptor and donor sequences
    – Stop codon
    – Predict alternative splicing...
    Computational gene prediction
    • De novo prediction – single genome
    – Trained with “typical” gene structures - learn exon-intron
    signals, translation initiation and termination signals e.g.
    Markov models
    – Many different predictions scored based on training set of
    known genes

    • Multiple genome
    – Compare confirmed gene sequences from other species
    – Coding regions more highly conserved  conservation
    indicates gene position
    – Pattern searching: Higher mutation rate of bases separated
    in multiples of three (mutations in 3rd position of codons are
    often silent)

    • Experimental data also contribute to many genome


    projects
    Brent, Nat Rev Genet. 2008 Jan;9(1):62-73

    • New methods weigh evidence from a variety of


    sources
    – Attempting to reproduce how a human annotator would
    work
    Experimental corroboration of models
    • Expressed Sequence Tags
    – Simple to obtain large volumes of data – sequence
    randomly from cDNA libraries
    – Problems:
    • Data sets can contain unprocessed transcripts (do not always
    confirm splicing)
    • Rarely cover 5’ end of gene
    • Generally “low-quality” sequences
    • High-throughput sequencing
    – “Next-generation” sequencers capable of directly
    sequencing mRNA
    – Likely to become more widely used in the future
    • Proteome data (peptide sequence data)
    How good are gene models?
    • Plasmodium falciparum (causative agent malaria)
    – genome sequenced in 2002, undergone considerable
    curation of gene models
    • Recent article: cDNA study of P. falciparum
    • Suggests ~25% of genes in Plasmodium
    falciparum are incorrect (85 genes out of 356
    sampled)
    • Majority of errors are in splice junctions (intron-
    exon boundaries)
    • What does this mean for other genomes...?
    – Likely that high percentage of gene sequences are
    incorrect!

    BMC Genomics. 2007 Jul 27;8:255.


    Proteome data for genome annotation

    • Motivation for genome annotation:


    – Can rule out that transcripts are non protein-coding
    – Large volumes of proteome data often collected for other
    purposes
    – Certain types of proteome data able to confirm the start
    codon of genes (difficult by other methods)
    – Even where considerable ESTs / cDNA sequencing has
    been performed, proteins can be detected with no
    corresponding EST evidence
    Proteogenomic study of Toxoplasma gondii

    • Proteome study of Toxoplasma gondii using


    three complementary techniques
    – parasite of clinical significance related to Plasmodium

    Study aims:
    • Identify as many components of the
    proteome as possible
    • Relate peptide sequence data back to
    genome to confirm genes
    • Relate protein expression data to
    transcriptional data (EST / microarray)
    Cut bands
    1D gel Trypsin digestion
    electrophoresis

    Mass spectrometry

    Cut gel spot Peptides


    Trypsin digestion

    2D gel electrophoresis
    Fractions
    Trypsin digestion

    Sequence database search


    (compare with theoretical spectra
    predicted for each peptide in DB)
    Liquid chromatography
    Database search strategy
    “Official” gene models
    Concatenate
    60MB genome databases
    sequence Alternative gene models
    predicted by gene
    finders Search all spectra

    ToxoDB

    Identify peptides and


    proteins
    ORFs predicted in a 6 frame
    translation

    Align peptide sequences back to corresponding genomic region

    = DNA sequence database

    = amino acid sequence database


    •Five exon gene; incomplete agreement between different gene models
    •Peptide evidence for all 5 exons and 2 introns out of 4
    •Note: Can only provide positive evidence, no peptides matched to 5’ and 3’
    termini of gene model
    -Appears to be additional exon at 5’
    -None of GLEAN, TwinScan or TigrScan algorithms appears to have made correct
    prediction
    50.m5694 sequence:
    MVEGVYSSFEAMIFSLPHACRTVTRT
    DLPSVKRFLTCVATSSKFPSESLGSIK
    SSFVSPFSRSSVQKPSSDKSINWNSDL
    FTFGTSML

    ORF/ part of TgGlimmerHMM sequence:


    VVGGFSSNFLSFFSVIITSVKMSDAEDVTFETA
    DAGASHTYPMQAGAIKKNGFVMLKGNPCKV
    VDYSTSKTGKHGHAKAHIVGLDIFTGKKYED
    VCPTSHNMEVPNVKRSEFQLIDLSDDGFCTLL
    LENGETKDDLMLPKDSEGNLDEVATQVKNLF
    TDGKSVLVTVLQACGKEKIIASKEL

    - All peptides matched to gene models on opposite strand


    Study outcomes
    • Protein evidence for approximately 1/3 of predicted
    genes (2250 proteins)
    • Around 2500 splicing events confirmed
    – Peptides aligned across intron-exon boundaries
    • Around 400 protein IDs appear to match alternative
    gene models
    • Genome database (ToxoDB) hosts peptide sequences
    aligned against gene models

    • Can we use informatics to improve this strategy...?

    Xia et al. (2008) Genome Biology,9(7),pp.R11


    Challenges of proteogenomics
    • Main informatics challenge:
    – A protein can usually only be identified if the gene sequence has
    been correctly predicted from the genome
    – In effect, would like to use MS data directly for gene discovery
    – But... searching a six frame genome translation is problematic

    • All peptide and protein identifications are probabilistic


    – False positive rate is proportional to search database size

    • On average only ~10-20% of spectra identify a peptide


    – Need methods that can exploit the rest of the meaningful spectra

    • When gene models change, protein identifications are out


    of date
    – No dynamic interaction between proteome and genome data
    Automated re-annotation pipeline
    Planned improvements to the informatics workflow:

    1. Re-querying pipeline
    – each time gene models change, all mass spectra are automatically re-
    queried
    2. Integrate peptide evidence directly into gene finding
    software
    3. Maximising the number of informative mass spectra
    4. Attempt to optimise algorithms for de novo sequencing of
    peptides
    5. N-terminal proteomics
    - Could be used to confirm gene initiation point
    Spectra

    Stage 1 Multiple
    Official
    database search Confirmed official
    gene set model
    engines
    Genome Gene
    sequence Finder
    Stage 2 Multiple
    Alternative Promote alternative
    gene models database search
    model
    engines

    Stage 3
    Modified de
    novo Novel ORF, splice
    junction
    algorithms
    Proteomic evidence

    • Spectra searched in series


    • Peptide evidence confirming official gene, alternative model, new ORF:
    • Direct flow back to modified gene finder
    • Produce new set of predictions
    • Iteratively improve number of spectra identified
    • In each iteration, fewer spectra flow on to stage 2 and 3
    Combining evidence in gene finders
    • Dynamically checking proposed gene models against peptide evidence
    • Combining evidence from different gene finding algorithms
    •In this case, probably no single algorithm appears to have correct model
    Query spectra using different search engines

    Peptide identifications

    Omssa
    Omssa X!Tandem
    Peptides

    X!Tandem Rescoring
    Combined
    Peptides Algorithm list
    (FDR)
    Mascot
    Peptides

    Mascot

    • Each search engine produces a different non-standard score of the quality of a match
    • Developed a search engine independent score, based on analysis of false discovery rate
    • Identifications made more search engines are scored more highly
    • Can generate 35% more peptide identification than best single search engine

    Jones et al. Improving sensitivity in proteome studies by analysis of false discovery


    rates for multiple search engines. PROTEOMICS, in press (2008)
    Conclusions

    • Proteome data is able to confirm gene models are


    correct
    – Currently data under-exploited
    • Challenges searching mass spec data directly against
    the genome for gene discovery
    • Build re-querying pipeline
    – Iteratively improve gene models
    – Improve capabilities for using multiple search engines
    – Integrate peptide evidence directly into gene finders
    Acknowledgments
    • Data from Wastling lab:
    – Dong Xia, Sanya Sanderson, Jonathan Wastling
    • ToxoDB at Upenn
    – David Roos, Brian Brunk

    Email: [email protected]

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