Gene prediction in flies

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Gene prediction in flies

• Background
• Gene prediction pipeline
• Resources

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Background
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Genome quality
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Genes in Drosophila melanogaster

• high gene density
• at least 20 with alternative transripts
• can be nested
• on the same strand
• on different strands
• di-cistronic
• involve trans-splicing
• exons from a different strand

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Gene prediction pipeline

• Gene prediction by homology
• no ab-initio predictions
• not using genomic alignments
• TBLASTN/Genewise process
• quick genome scan to find putative gene
  containing regions
• aligning peptide sequence to genomic fragment
  using a gene model
• cds
• introns
• splice-sites

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(No Transcript)
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Sensitivity Selectivity - Speed

• Genome scan
• strict trade-off between
• sensitivity versus memory/time
• Transcript prediction
• t O(MN)
• N length of peptide sequence quite short
• M length of DNA sequence large
• you want to minimize
• the length of the genomic sequence to search
• the number of fragments you align

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Solutions

• ENSEMBL Minigenes
• cut out putative introns
• My pipeline
• priority lists
• gene structure conservation

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Difficulties

• Terminal exons
• short and thus alignment signal is weak
• Spindly genes
• there is no length penalty on introns

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Concepts

• Predict in three passes
• Predict clear cut cases
• Predict dubious cases
• only if they don't overlap with a previous
  prediction
• Predict alternative transcripts
• Iteratively search for duplications
• Accept a prediction with conserved exon boundaries

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Conservation of gene structure
Query
Conserved
Prediction
Query
Partially conserved
Prediction
Query
Single exon
Prediction
Query
Retrotransposed
Prediction
Query
Unconserved
Prediction
(exon boundaries of query/prediction mapped on
query protein)
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Quality control

• Classify predictions into categories
• Full length or fragment
• Gene or pseudogene
• Conserved or not conserved gene structure
• Heuristically remove predictions
• that are redundant
• that are in conflict
• nested genes
• good predictions take precedence over bad
  predictions

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Results

• http//wwwfgu.anat.ox.ac.uk8080/cgi-bin/gbrowse

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Number of predicted genes
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Orthology assignments

• Genes in D. melanogaster with ortholgs

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Technical details

• Hardware
• 28 dual CPU nodes with 2Gb memory
• sun grid engine (SGE)
• Pipeline logic
• gmake
• Tasks
• Python scripts (and Perl scripts)
• Bash/awk scripts
• Database
• Postgres

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Downstream analysis

• Pairwise orthology assignment
• PhyOP Pipeline (Leo Goodstadt (2006))
• Multiple orthology assignment
• My own concoction based on graph clustering with
  some consistency criteria
• Multiple alignment of cds
• Dialign (lt50 sequences)
• Muscle (lt500 sequences)

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Phylogenetic analysis

• 14,000 GBlocks cleaned multiple alignments
• Calculation of ka and ks with PAML
• Phylogenetic trees
• Genome trees
• Gene trees
• built with Fitch/Kitsch

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Odds and bits

• Mapping of Pdb -gt Uniprot -gt dmel proteins
• Mapping of Interpro domains onto predictions
• not up-to-date
• Codon bias analysis
• ENC, CAI, information theoretic measures
• GC3, GC3_4D

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Comparison of measures
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Other groups

• see http//rana.lbl.gov/drosophila/wiki/index.php/
  Main_Page
• Gene predictions by others
• Don Gilbert SNAP
• Lior Pachter GeneMapper (genomic alignments)
• Eisen Lab TBLastN Genewise/Exonerate,
  GeneMapper
• Batzoglou Lab CONTRAST
• Brent Lab N-Scan
• Guigo geneid and SGP2

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http//insects.eugenes.org/species/news/genome-sum
maries/genepredictions.html
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Consensus predictions

• Gbrowser comparison of all gene predictions
• http//rana.lbl.gov/drosophila/gbrowse/cgi-bin/gbr
  owse
• Mike Eisen's group GLEAN consensus set
• Don Gilbert http//insects.eugenes.org/species/
• Other resources
• tRNA predictions
• genome alignments