CGPDB Presentation

1
Linear graphical representation of BLAST search
of Arabidopsis genome against Lettuce/Sunflower
EST assemblies.
http//cgpdb.ucdavis.edu/database/est_vs_ath/tigr_
vs_let_and_sun.html
Lettuce/Sunflower EST CGPDB project. Data
analysis, assembly visualization and
validation. Alexander Kozik, Brian Chan, Richard
Michelmore. Department of Vegetable Crops,
University of California at Davis, CA 95616.
Linear graphical representation of BLAST search
against the Arabidopsis genome. Each element
represents a 'gene' - predicted ORF (TIGR
version, September 2002). Elements are ordered
according to position on chromosome and are web
links to corresponding entries in the CGP
database. Color intensity indicates level of
similarity (normalized Expectation values
-log(Exp)). Green - significant hit to lettuce,
Red - significant hit to sunflower. Yellow -
significant hit to both. White blocks separate
the Arabidopsis chromosomes.
Over 60,000 lettuce and 40,000 sunflower ESTs
from multiple libraries have been assembled using
the CAP3 program (http//genome.cs.mtu.edu/cap/cap
3.html) and organized into the Compositae Genome
Project database (http//cgpdb.ucdavis.edu/).
This assembly represents about 19,000 lettuce and
12,000 sunflower unigenes. mySQL
(http//www.mysql.com/) was chosen as an
efficient tool to manage the data. Custom PHP and
Python programs were developed with publicly
available php_my_admin software to manipulate the
data and visualize the assemblies. To exploit the
generation of the ESTs from different genotypes
representing mapping parents of lettuce and
sunflower, we developed a new software to
identify possible polymorphisms. About 250
insertions/deletions (INDELs) and 2,500
substitutions (SNPs) have been discovered for
lettuce and sunflower assemblies using custom
Python scripts. Wet lab experiments have
confirmed the predicted polymorphism in 90
cases. A new clustering algorithm was used to
find putative COS (conserved ortholog set)
markers. About 1,200 lettuce and 500 sunflower
putative COS markers have been identified based
on clustering analysis with the complete
Arabidopsis genome. EST assemblies have been
analyzed for multidomain proteins, possible
chimeric clones and misassembled contigs using
graph theory and our custom Graph9 program.
Clusters of multigene families have been
visualized using PhyloGrapher program
(http//cgpdb.ucdavis.edu/PhyloGrapher/).
Image created with PyMood (http//www.pymood.com/
)
Sequence clustering finding chimeric and
multidomain ESTs
Scheme of Data Processing and SNP/INDEL Discovery
Pipeline
Two different genotypes for each genus (Lettuce
cv. Salinas and L. serriola) (Sunflower RHA801
and RHA280)
chimeric sequence
cDNA library construction (individual libraries
for each genotype) Sequencing
Raw Chromatograms (reads) processing by
Phred-CrossMatch
Clustering visualized by PhyloGrapher, for
details see http//www.atgc.org/
Individual CAP3 assembly for each
genus different genotypes analyzed together
Clustering analysis by Graph9 program BLAST EST
assembly against itself --gt --gt Generation of
"Matrix" file using tcl_blast_parser.tcl program
--gt --gt Clustering and bridges search by
Graph9 program.
Processing of the CAP3 output with custom Python
scripts and generation of tab-delimited files
ready to go into relational mySQL database
Finding in the assembly all mismatches in
individual sequences versus consensus sequence.
If all mismatches for given position belong to
one genotype it is considered as a potential
polymorphic site (SNP or INDEL)
Graph9 output with bridges info, see table
lettuce_clustering at CGPDB http//cgpdb.ucdavis.e
du/ for details
Conserved Ortholog Set (COS) Markers candidates
Contig Viewer
http//cgpdb.ucdavis.edu/database/chromat_viewer/C
ontigViewer_MMX.php
Pipeline to process BLAST output Blast parser
generates "Matrix" file form regular BLAST
output. Graph9 program analyzes "Matrix" file
and generates "Group Degree Info" file. "Group
Degree Info" file contains full information about
sequence clustering based on "Matrix" file. See
http//cgpdb.ucdavis.edu/BlastParser/Blast_Parser.
html
on-line Contig Viewer is a set of PHP scripts to
navigate assembly in full details. Contig Viewer
displays information about assembly, highlights
sites of polymorphism, provides web links to
BLAST reports for consensus and individual
sequences. All underlying data are stored in
mySQL database. There are four tables that
provide full information to display assembly
graphically. All tables were derived by
processing of CAP3 output by custom Python
scripts.
Example of false single hit
Strategy to identify COS candidates Clustering
analysis using Graph9 program and removing from
potential COS set all EST-Arabidopsis clusters
with multiple Arabidopsis nodes. Clustering
parameters were Expect cutoff 1e-10, Identity
cutoff 20 and Overlap cutoff 50 amino acids.
Table with overlap info for every sequence in the
assembly
Table with CAP3 clip info for every sequence
Table with mismatch info sequences vs consensus
of the assembly
CAP3 assembly output files are sufficient to
extract full information about
polymorphic sites. Besides numerical
information, CGPDB provides full access
to raw chromatograms for every
sequence in the database. Therefore base
calling can be verified for every
nucleotide in lettuce/sunflower
ESTs
Graphical representation of BLAST search lettuce,
sunflower, tomato and corn ESTs against
Arabidopsis genome. Potential conserved
orthologs. Color scheme lettucesunflower -
green, tomato - red, corn - blue. Additive color
mixing reflects EST representation for
Arabidopsis gene (ORF). white red green
blue, yellow red green, cyan green blue,
purple red blue. Genes are web links to
corresponding entries in CGP database
(http//cgpdb.ucdavis.edu/database/est_vs_ath/arab
idopsis_cos_map.html)
Table with tissue info for every sequence