1. Tomato genome annotation pipeline in Cyrille2
Erwin Datema

2. Contents of the annotation pipeline
Annotation on the BAC level
Gene prediction
Repeat identification
Other features
Annotation on the gene level (work in progress)
blastx vs NCBI’s nr (sequence similarity)
InterProScan (domain identifcation)

3. Ab initio gene structure prediction
Ab initio predictors included in the pipeline
Genscan
GlimmerHMM (trained on tomato!)
GeneId (has been trained on Solanaceae)
SNAP
Augustus (predicts alternative spliced variants)

4. Alignment-based gene structure prediction (1)
Transcript alignment (blastn + Sim4)
SGN tomato UniGenes ( UniGenes)
SGN potato UniGenes ( UniGenes)
SGN coffee UniGenes ( UniGenes)
SGN pepper UniGenes (9.554 UniGenes)
SGN petunia Unigenes (5.135 UniGenes)
SGN S. melongena UniGenes (1.841 UniGenes)
NCBI full-length tomato cDNAs (678 cDNAs)
Protein alignment (tblastn + GeneWise)
TAIR6 Arabidopsis thaliana proteome ( proteins)
TIGR4 Oryza sativa proteome ( proteins)
UniProt Plant division ( proteins)

5. Additional feature prediction
Repeat Identification
Tandem Repeats Finder
RepeatMasker
RepBase + ‘default’ features (low complexity, etc)
TIGR Solanum lycopersicon repeat library V2
SGN Solanum lycopersicon UniRepeats
Feature prediction
tRNAscan-SE
MarScan
GeneSplicer
Marker identification (blastn + Sim4)

6. Preliminary results Annotation of chromosome 6 BACs phase 1, 2 and 3
632 contigs
Older version of the pipeline
GlimmerHMM only trained on Arabidopsis
2 UniGene sets (tomato, potato)
2 protein sets (Arabidopsis, UniProt plant)
Protein alignment parameters too strict

7. The genomic landscape of chromosome 6
632 contigs have been annotated
Length of contigs varies between 348 – nt
Average length of nt, median length of nt
Total length of nt
GC content: 29.9% min, 34.1% avg, 42.2% max (sequences longer than nt)

8. Ab initio gene prediction
Note: Augustus predictions include up to 3 splice variants per gene
Estimated gene density is 1 gene per 5 kb
~1.200 genes in currently sequenced BACs

9. Transcript alignment-based gene prediction
Tomato
UniGenes (derived from ESTs)
574 hits to the contigs
Potato
UniGenes (derived from ESTs)
631 hits to the contigs

10. Protein alignment-based gene prediction
UniProt Plant proteins
protein sequences from the plant kingdom
195 hits to the contigs
Arabidopsis thaliana TAIR6 annotation
protein sequences
228 hits to the contigs

11. Repeat density TIGR Tomato Repeat Library (95 repeats)
118 regions spanning nt
Minimum 48 nt, average 449 nt, maximum nt
SGN Tomato UniRepeats (668 repeats)
2.860 regions spanning nt
Minimum 10 nt, average 427 nt, maximum nt
Tandem repeats
1.313 regions spanning nt
Minimum 24 nt, average 120 nt, maximum nt

12. Additional features 74 markers could be aligned
alignment quality unverified
39 predicted tRNA genes
1.301 predicted MAR/SAR elements

13. Generic Genome Browser (1)

14. Generic Genome Browser (2)

15. Generic Genome Browser (3)

16. Recent work GeneModelCollector JIGSAW
Tries to find ‘full’ open reading frames in aligned UniGenes
Automatic generation of gene predictor training set
Parameters?
JIGSAW
Appears not to provide a prediction for every region which contains annotations
Training?

17. Future Work – Tomato Annotation Pipeline
Gene prediction
Combining predictions into a single consensus model
Train individual predictors with recently curated tomato gene set
Automated functional annotation of genes
“Giving a biological meaning to the nicely colored bars”
blastx
InterProScan

18. Future Work – Tomato Genome Browser
Annotation of features
Meaningful names for features such as genes, marker alignments, blast hits
More detailed and better readable data when clicking on a feature
Links to external data sources
NCBI GenBank
SGN

19. Acknowledgements Cyrille2 development
Mark Fiers
Ate van der Burgt
Joost de Groot
Tomato BAC sequencing (chromosome 6)
Greenomics
Supervision
Willem Stiekema
Roeland van Ham