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: Andrew.jones@liv.ac.uk