Eukaryotic Gene Finding Adapted in part from http://online.itp.ucsb.edu/online/infobio01/burge/
Prokaryotic vs. Eukaryotic Genes Prokaryotes small genomes high gene density no introns (or splicing) no RNA processing similar promoters overlapping genes Eukaryotes large genomes low gene density introns (splicing) RNA processing heterogeneous promoters polyadenylation
Pre-mRNA Splicing ... ... U 1 s n R N P 2 intronic repressor 5 ’ splice signal U 2 A F 6 5 3 1 s n R N P SR proteins intron definition exon definition exonic enhancers 5 ’ splice signal 3 polyY branch signal intronic enhancers exonic repressor ... (assembly of spliceosome, catalysis) ...
Some Statistics On average, a vertebrate gene is about 30KB long Coding region takes about 1KB Exon sizes can vary from double digit numbers to kilobases An average 5’ UTR is about 750 bp An average 3’UTR is about 450 bp but both can be much longer.
Human Splice Signal Motifs
Semi-Markov HMM Model
Genscan HSMM
GenScan States N - intergenic region P - promoter F - 5’ untranslated region Esngl – single exon (intronless) (translation start -> stop codon) Einit – initial exon (translation start -> donor splice site) Ek – phase k internal exon (acceptor splice site -> donor splice site) Eterm – terminal exon (acceptor splice site -> stop codon) Ik – phase k intron: 0 – between codons; 1 – after the first base of a codon; 2 – after the second base of a codon
GenScan features Model both strands at once Each state may output a string of symbols (according to some probability distribution). Explicit intron/exon length modeling Advanced splice site modeling Parameters learned from annotated genes Separate parameter training for different CpG content groups
GenScan Signal Modeling PSSM: P(S) = P1(S1)•P2(S2) •…•Pn(Sn) PolyA signal Translation initiation/termination signal Promoters WAM: P(S) = P1(S1) •P2(S2|S1)•…•Pn(Sn|Sn-1) 5’ and 3’ splice sites
HMM-based Gene Finding GENSCAN (Burge 1997) FGENESH (Solovyev 1997) HMMgene (Krogh 1997) GENIE (Kulp 1996) GENMARK (Borodovsky & McIninch 1993) VEIL (Henderson, Salzberg, & Fasman 1997)
GenomeScan proteins are available. Idea: We can enhance our gene prediction by using external information: DNA regions with homology to known proteins are more likely to be coding exons. Combine probabilistic ‘extrinsic’ information (BLAST hits) with a probabilistic model of gene structure/composition (GenScan) Focus on ‘typical case’ when homologous but not identical proteins are available.
GeneWise [Birney, Amitai] Motivation: Use good DB of protein world (PFAM) to help us annotate genomic DNA GeneWise algorithm aligns a profile HMM directly to the DNA
Sample GeneWise Output
Developing GeneWise Model Start with a PFAM domain HMM Replace AA emissions with codon emissions Allow for sequencing errors (deletions/insertions) Add a 3-state intron model
GeneWise Model
GeneWise Intron Model PY tract central spacer 5’ site 3’ site
GeneWise Model Viterbi algorithm -> “best” alignment of DNA to protein domain Alignment gives exact exon-intron boundaries Parameters learned from species-specific statistics
GeneWise problems Only provides partial prediction, and only where the homology lies Does not find “more” genes Pseudogenes, Retrotransposons picked up CPU intensive Solution: Pre-filter with BLAST
Summary Genes are complex structures which are difficult to predict with the required level of accuracy/confidence Different approaches to gene finding: Ab Initio : GenScan Ab Initio modified by BLAST homologies: GenomeScan Homology guided: GeneWise