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Expressed Sequence Tag (EST)
Vassilos Ioannidis - 2004
(modified from Lorenzo Cerutti, Victor Jongeneel, Anne Estreicher, )
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ESTs - outline Introduction
- Introduction
- Improving ESTs
- pre-processing
- clustering
- assembling
- Gene indices / UniGene & TIGR db
- Practical example
- Concluding Remarks
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Traditional sequencing
cDNA clones isolated on the basis of some functional property of interest
to a group
EST sequencing
Large-scale sampling of end sequences of all cDNA clones present in a
library
Full-length sequencing
Systematic attempts to obtain high-quality sequences of cDNA clones
representing all transcribed genes
Transcriptome sequencing Introduction
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What are ESTs Introduction
cDNA libraries prepared from various organisms, tissues and cell lines
using directional cloning
Gridding of individual clones using robots
For each clone, single-pass sequencing of both ends (5 and/or 3) of insert
Deposit readable part of sequence in database
ESTs represent partial sequences of cDNA clones (300 bp -> 700 bp)
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What are ESTs Introduction
mRNA
AAAAA
mRNA
cDNA
AAAAA
Synthesis of 1 strand of DNA
(Reverse Transcriptase)
cDNA
cDNA
RNA degradation
Synthesis of 2 strand of DNA
(DNA Polymerase)
Cloning vector
MCS
5 3
3 5
T3
T7
Cloning &
Sequencing
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Why EST sequencing? Introduction
Fast & cheap (almost all steps are automated)
They represent the most extensive available survey of the transcribed portion
of genomes.
There are indispensable for gene structure prediction, gene discovery and
genome mapping:
-> provide experimental evidence for the position of exons
-> provide regions coding for potentially new proteins
-> characterization of splice variants and alternative polyadenilation
Provide an alternative to library screening
-> short tag can lead to a cDNA clone
Provide an alternative to full-length cDNA sequencing
-> sequences of multiple ESTs can reconstitute a full-length cDNA
Single Nucleotide Polymorphism (SNP) data mining
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Most are native, meaning that clone frequency reflects mRNA
abundance
Most are primed with oligo(dT), meaning that 3 ends are heavily
represented
The complexity of libraries is extremely variable
Normalized libraries are used to enrich for rare mRNAs
cDNA libraries Introduction
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cDNA libraries used Introduction
Large number of libraries represented
Most libraries managed by the IMAGE consortium (http://image.llnl.gov/)
Human & mouse libraries are the most abundantly represented:
Many tissues still not sampled
Quality very uneven
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EST databases Introduction
The data sources for clustering can be in-house, proprietary, public database or
a hybrid of this (chromatograms and/or sequence files).
Each EST must have the following information:
A sequence ID (ex. sequence-run ID)
Location in respect of the poly A (3' or 5')
The CLONE ID from which the EST has been generated
Organism
Tissue and/or conditions
The sequence
The EST can be stored in FASTA format:
>T27784 EST16067 Human Endothelial cells Homo sapiens cDNA 5'
CCCCCGTCTCTTTAAAAATATATATATTTTAAATATACTTAAATATATATTTCTAATATC
TTTAAATATATATATATATTTNAAAGACCAATTTATGGGAGANTTGCACACAGATGTGAA
ATGAATGTAATCTAATAGANGCCTAATCAGCCCACCATGTTCTCCACTGAAAAATCCTCT
TTCTTTGGGGTTTTTCTTTCTTTCTTTTT
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EST databases Introduction
Public EST databases
EMBL/GenBank have separate sections for EST sequences
ESTs are the most abundant entries in the databases (>60%)
ESTs are now separated by division in the databases:
-> human, mouse, plant, prokaryote, (EMBL)
ESTs sequences are submitted in bulk, but do have to meet minimal quality
criteria (Phred score >20%, ie <1% error)
Private EST databases
(producing and selling access to EST data has proven to be a lucrative business)
Human Genome Sciences (http://www.hgsi.com/) exploit the data itself, and get
patents on promising genes found in its databases
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EST / EST databases quality Introduction
ESTs represent partial sequences of cDNA clones (300 bp -> 700 bp)
-> No attempt to obtain the complete sequence (no overlap necessary)
-> A single EST represents only a partial gene sequence
-> Not a defined gene/protein product
Single, unverified runs from the 5 and/or 3 ends of cDNA clones
-> high error rates (~1/100)
-> frequent sequence compression and frame-shift errors
Trivial contaminants are common (vector, rRNA, mitRNA, )
Not curated in a highly annotated form
High redundancy in the data (native databases: clone frequency reflects mRNA abundance)
Databases are skewed for sequences near 3-end of mRNAs (normalization)
For most ESTs, no indication as to the gene from which they are derived
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Clone availability Introduction
In principle, all clones produced by IMAGE are publicly available
Distributors:
- US: ATCC (http://www.lgcpromochem.com/atcc/) and Invitrogen
(http://clones.invitrogen.com/cloneinfo.php?clone=est)
- UK: HGMP (http://www.hgmp.mrc.ac.uk/geneservice/reagents/index.shtml)
- D: RZPD (http://www.rzpd.de/products/clones/)
Notice:
- Error rate is high: ~30% chance that clone doesnt have expected sequence
- Invitrogen sells sets of sequence verified clones
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EST entry in EMBL Introduction
ID AI242177 standard; RNA; EST; 581 BP.
AC AI242177;
SV AI242177.1
DT 05-NOV-1998 (Rel. 57, Created)
DT 03-MAR-2000 (Rel. 63, Last updated, Version 3)
DE qh81g08.x1 Soares_fetal_liver_spleen_1NFLS_S1 Homo sapiens cDNA
DE clone IMAGE:1851134 3' similar to gb:M10988 TUMOR NECROSIS FACTOR
DE PRECURSOR (HUMAN);, mRNA sequence.
RN [1]
RP 1-581
RA NCI-CGAP;
RT National Cancer Institute, Cancer Genome Anatomy Project (CGAP), Tumor
RT Gene Index http://www.ncbi.nlm.nih.gov/ncicgap;
RL Unpublished.
DR RZPD; IMAGp998P154529; IMAGp998P154529.
CC On May 19, 1998 this sequence version replaced gi:2846208.
CC Contact: Robert Strausberg, Ph.D.
CC Tel: (301) 496-1550
CC Email: [email protected]
CC This clone is available royalty-free through LLNL ; contact the
CC IMAGE Consortium ([email protected]) for further information.
CC Insert Length: 1280 Std Error: 0.00
CC Seq primer: -40UP from Gibco
CC High quality sequence stop: 463.
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EST entry in EMBL Introduction
FH Key Location/Qualifiers
FH
FT source 1..581
FT /db_xref=taxon:9606
FT /db_xref=ESTLIB:452
FT /db_xref=RZPD:IMAGp998P154529
FT /note=Organ: Liver and Spleen; Vector: pT7T3D (Pharmacia)
FT with a modified polylinker; Site_1: Pac I; Site_2: Eco RI;
FT This is a subtracted version of the original Soares fetal
FT liver spleen 1NFLS library. 1st strand cDNA was primed
FT with a Pac I - oligo(dT) primer [5'
FT AACTGGAAGAATTAATTAAAGATCTTTTTTTTTTTTTTTTTTT 3'],
FT double-stranded cDNA was ligated to Eco RI adaptors
FT (Pharmacia), digested with Pac I and cloned into the Pac I
FT and Eco RI sites of the modified pT7T3 vector. Library
FT went through one round of normalization. Library
FT constructed by Bento Soares and M.Fatima Bonaldo.
FT /sex=male
FT /organism=Homo sapiens
FT /clone=IMAGE:1851134
FT /clone_lib=Soares_fetal_liver_spleen_1NFLS_S1
FT /dev_stage=20 week-post conception fetus
FT /lab_host=DH10B (ampicillin resistant)
SQ Sequence 581 BP; 179 A; 130 C; 135 G; 137 T; 0 other;
cttttctaag caaactttat ttctcgccac tgaatagtag ggcgattaca gacacaactc 60

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From an EST entry in EMBL to clone shopping
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Improving ESTs Introduction
The value of ESTs can be greatly enhanced by
Pre-processing
(Steps required to clean & prepare ESTs sequences)
Clustering
(minimization of the chance to cluster unrelated sequences)
Assembling
(derive consensus sequences from overlapping ESTs belonging to the same cluster)
Mapping
(associate ESTs or ESTs contigs with exons in genomic sequences)
Interpreting
(find and correct coding regions)
in order to :
-> solve redundancy & help correcting errors
-> get longer & better annotated sequences
-> allow easier association to mRNAs & proteins
-> allow detection of splice variants
-> fewer sequences to analyze
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Improving ESTs Pre-processing
EST pre-processing consists in a number of essential steps to minimize the
chance to cluster unrelated sequences:
Screening out low quality regions:
- Low quality sequence readings are error prone
Screening out contaminations (rRNA, mitRNA, )
Screening out vector sequences (vector clipping)
Screening out repeat sequences (repeat masking)
Screening out low complexity sequences
Softwares:
Phred (Ewig et al., 1998)
- Reads chromatograms and assesses a quality value to each nucleotide
VecScreen (http://www.ncbi.nlm.nih.gov/VecScreen)
RepeatMasker (http://ftp.genome.washington.edu/RM/RepeatMasker.html)

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Improving ESTs Pre-processing
Vector clipping and contaminations
Vector sequences can skew clustering even if a small vector fragment remains
in each read. Therefore vector sequences must be removed:
- Delete 5 and 3 regions corresponding to the vector used for cloning
- Detection of vector sequences is not a trivial task, because they usually
lie in the low quality region of the sequence
- UniVec is a non-redundant vector database available from the NCBI
(http://www.ncbi.nlm.nih.gov/VecScreen/UniVec.html)
Contaminations can also skew clustering and therefore must be removed:
- Find and delete bacterial DNA, yeast DNA,
Standard pairwise alignment programs are used for the detection of vector
sequences and other contaminants (cross-match, BLASTN, FASTA, )
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Improving ESTs Pre-processing
Repeats masking
Some repetitive elements found in the human genome:
LINEs (long interspersed elements) 6-8 kb 850000 21%
SINEs (short interspersed elements) 100-300 bp 1500000 13%
_______________________________________________________________
Length Copy number
Fraction of
the genome
LTR (autonomous) 6-11 kb
LTR (non-autonomous) 1.5-3 kb
450000 8%
DNA transposons (autonomous) 2-3 kb
DNA transposons (non-autonomous) 80-3000 bp
300000 3%
SSRs (simple sequence repeats or micro satellites and mini satellites) 3%
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Improving ESTs Pre-processing
Repeats masking
Repeated elements:
- They represent a big part of the mammalian genome
- They are found in a number of genomes (plants, )
- They induce errors in clustering and assembling
- They should be MASKED, not deleted, to avoid false sequence assembling
(also interesting for evolutionary studies. SSRs important for mapping of diseases)
Tools to find repeats:
- RepeatMasker has been developed to find repetitive elements and low-
complexity sequences. It uses the cross-match program for the pairwise
alignments (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker)
- MaskerAid improves the speed of RepeatMasker by ~30 folds using WU-
BLAST instead of cross-match (http://sapiens.wustl.edu/maskeraid)
- RepBase is a database of prototypic sequences representing repetitive DNA
from different eukaryotic species (http://www.girinst.org/Repbase_Update.html)
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Improving ESTs Pre-processing
Low complexity masking
Low complexity sequences contain an important bias in their nucleotide
compositions (poly A tracts, AT repeats, etc.)
Low complexity regions can provide an artifactual basis for cluster membership
Clustering strategies employing alignable similarity in their first pass are very
sensitive to low complexity sequences
Some clustering strategies are insensitive to low complexity sequences, because
they weight sequences in respect to their information content (ex. d2-cluster).
Programs as DUST (NCBI) can be used to mask low complexity regions
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Improving ESTs Pre-processing
ATGAATGTAATCTAATAGANGCCTAATCAGCCCACCATGTTCTCCACTGAAAAATCCTCT
CCCCCGTCTCTTTAAAAATATATATATTTTAAATATACTTAAATATATATTTCTAATATC
TTTAAATATATATATATATTTNAAAGACCAATTTATGGGAGANTTGCACACAGATGTGAA
TTCTTTGGGGTTTTTCTTTCTTTCTTTTTTGATTTTGCACTGGACGGTGACGTCAGCCAT
GTACAGGATCCACAGGGGTGGTGTCAAATGCTATTGAAATTNTGTTGAATTGTATACTTT
TTCACTTTTTGATAATTAACCATGTAAAAAATGAACGCTACTACTATAGTAGAATTGAT
Base calling
Select high quality reads
Vector clipping
CCCCCGTCTCTTTAAAAATATATATATTTTAAATATACTTAAATATATATTTCTAATATC
TTTAAATATATATATATATTTNAAAGACCAATTTATGGGAGANTTGCACACAGATGTGAA
ATGAATGTAATCTAATAGANGCCTAATCAGCCCACCATGTTCTCCACTGAAAAATCCTCT
TTCTTTGGGGTTTTTCTTTCTTTCTTTTTTGATTTTGCACTGGACGGTGACGTCAGCCAT
GTACAGGATCCACAGGGGTGGTGTCAAATGCTATTGAAATTNTGTTGAATTGTATACTTT
TTCACTTTTTGATAATTAACCATGTAAAAAATGXXXXXXXXXXXXXXXXXXXXXXXXXX
Repeat/Low complexity masking
CCCCCGTCTCTTTAAAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNTTNAAAGACCAATTTATGGGAGANTTGCACACAGATGTGAA
ATGAATGTAATCTAATAGANGCCTAATCAGCCCACCATGTTCTCCACTGAAAAATCCTCT
TTCTTTGGGGTTTTTCTTTCTTTCTTTTTTGATTTTGCACTGGACGGTGACGTCAGCCAT
GTACAGGATCCACAGGGGTGGTGTCAAATGCTATTGAAATTNTGTTGAATTGTATACTTT
TTCACTTTTTGATAATTAACCATGTAAAAAATGXXXXXXXXXXXXXXXXXXXXXXXXXX
Sequence ready for clustering
CCCCCGTCTCTTTAAAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNTTNAAAGACCAATTTATGGGAGANTTGCACACAGATGTGAA
ATGAATGTAATCTAATAGANGCCTAATCAGCCCACCATGTTCTCCACTGAAAAATCCTCT
TTCTTTGGGGTTTTTCTTTCTTTCTTTTTTGATTTTGCACTGGACGGTGACGTCAGCCAT
GTACAGGATCCACAGGGGTGGTGTCAAATGCTATTGAAATTNTGTTGAATTGTATACTTT
TTCACTTTTTGATAATTAACCATGTAAAAAATG
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Improving ESTs Clustering
EST clustering consists in incorporating overlapping ESTs which tag the same
Transcript of the same gene in a single cluster
For clustering, we measure the similarity (distance) between any 2 sequences.
The distance is then reduced to a simple binary value:
- accept or reject two sequences in the same cluster
Similarity can be measured using different algorithms:
Pairwise alignment algorithms:
Smith-Waterman is the most sensitive, but time consuming (ex. cross-match);
Heuristic algorithms, as BLAST and FASTA, trade some sensitivity for speed.
Non-alignment based scoring methods:
d2-cluster algorithm: based on word comparison and composition (word identity
and multiplicity) (Burke et al., 99). No alignments are performed ) fast.
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Improving ESTs Clustering
Stringent clustering:
Greater initial fidelity
One pass
Lower coverage of expressed gene data
Lower cluster inclusion of expressed gene forms
Shorter consensi
Loose clustering:
Lower initial fidelity
Multi-pass
Greater coverage of expressed gene data
Greater cluster inclusion of alternate expressed forms
Longer consensi
Risk to include paralogs in the same gene index
TIGR
UniGene
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Improving ESTs Clustering
Supervised clustering
ESTs are classified with respect to known reference sequences or "seeds" (full
length mRNAs, exon constructs from genomic sequences, previously assembled
EST cluster consensus)
Unsupervised clustering
ESTs are classified without any prior knowledge (ab initio)
The two major gene indices use different EST clustering methods:
TIGR Gene Index uses a stringent and supervised clustering method, which
generates shorter consensus sequences and separates splice variants
A combination of supervised and unsupervised methods with variable levels of
stringency is used in UniGene. No consensus sequences are produced
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Improving ESTs Assembling
Assembling, processing and cluster joining
A multiple alignment for each cluster can be built (assembly) and consensus
sequences generated (processing)
A number of program are available for assembly and processing:
- PHRAP (http://www.phrap.org/)
- TIGR ASSEMBLER (Sutton et al., 95)
-
Assembly and processing result in the production of consensus sequences
and singletons.
Consensus sequences are useful:
- to help visualizing splice variants;
- to reduce the size of data to analyze;
- for gene structure;
- ...
UniGene
TIGR
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Improving ESTs Assembling
Assembly &
Processing
Joining
Assembling, processing and cluster joining
All ESTs generated from the same cDNA clone correspond to a single gene
Generally the original cDNA clone information is available (~90%)
Using the cDNA clone information and the 5 and 3 reads information,
clusters can be joined
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The need for a gene index U&T db
- All high-throughput biology methods require a unique and reliable way to
describe the genes they are analyzing
- This index should be stable, unique, extensible, and independent of a
system of nomenclature
- The index should document all transcript sequences belonging to the
corresponding gene
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Some commonly used gene indices U&T db
EMBL/GenBank/DDBJ accession numbers
Unique and universally accepted BUT
Highly redundant (many entries per gene)
Unigene cluster identifiers (NCBI)
Widely used and non-redundant BUT
Rely on clustering procedure (unreliable) AND
Unstable clusters change with each build
RefSeq accession numbers (NCBI)
Stable and non-redundant BUT
Still very far from comprehensive AND
Many RefSeq sequences are incomplete AND
Splice variants are not systematically documented
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Indices: The Unigene database U&T db
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Indices: The Unigene database U&T db
Unigene (http://www.ncbi.nlm.nih.gov/UniGene/) is an ongoing effort at NCBI to
cluster EST sequences with traditional gene sequences
For each cluster, there is a lot of additional information included
(Represented organisms comprise animals & plants)
Unigene is regularly rebuilt. Therefore:
cluster identifiers are not stable gene indices !!!
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Indices: The Unigene database U&T db
UniGene procedure: (supervised or unsupervised, multipass)
Screen for contaminants, repeats, and low-complexity regions in GenBank:
- Low-complexity are detected using Dust
- Contaminants (vector, linker, bacterial, mitochondrial, ribosomal sequences)
are detected using pairwise alignment programs
- Repeat masking of repeated regions (RepeatMasker)
- Only sequences with at least 100 informative bases are accepted
Clustering procedure:
- Build clusters of genes and mRNAs (GenBank)
- Add ESTs to previous clusters (megablast)
- ESTs that join two clusters of genes/mRNAs are discarded
- Any resulting cluster without a polyadenilation signal or at least two 3' ESTs
is discarded (*)
- The resulting clusters are called anchored clusters since their 3' end is
supposed known
(*: UniGene rule)
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Indices: The Unigene database U&T db
UniGene procedure:
Ensures that the 5' and 3' ESTs from the same cDNA clone belongs to the
same cluster
ESTs that have not been clustered, are reprocessed with lower level of
stringency
ESTs added during this step are called guest members
Clusters of size 1 (containing a single sequence) are compared against the
rest of the clusters with a lower level of stringency and merged with the
cluster containing the most similar sequence
For each build of the database, clusters IDs change if clusters are split or
merged.
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Indices: The TIGR database U&T db
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Indices: The TIGR database U&T db
TIGR produces Gene Indices for a number of organisms (http://www.tigr.org/tdb/tgi).
TIGR Gene Indices are produced using stringent supervised clustering methods
Clusters are assembled in consensus sequences, called tentative consensus (TC)
sequences, that represent the underlying mRNA transcripts
The TIGR Gene Indices building method tightly groups highly related
sequences and discard under-represented, divergent, or noisy sequences
TIGR Gene Indices characteristics:
- separate closely related genes into distinct consensus sequences;
- separate splice variants into separate clusters;
- low level of contamination.
TC sequences can be used for genome annotation, genome mapping, and
identification of orthologs/paralogs genes
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Indices: The TIGR database U&T db
TIGR procedure: (supervised, stringent)
EST sequences recovered form dbEST (http://www.ncbi.nlm.nih.gov/dbEST);
Sequences are trimmed to remove:
vectors
polyA/T tails
adaptor sequences
bacterial sequences
Get expressed transcripts (ETs) from EGAD (http://www.tigr.org/tdb/egad/egad.shtml)
EGAD (Expressed Gene Anatomy Database) is based on mRNA and CDS (coding sequences)
from GenBank
Get TCs and singletons from previous database build
Supervised and strict clustering
Use ETs, TCs, and CDSs as seed;
Compare cleaned ESTs to the template using FLAST (a rapid pairwise comparison
program).
Sequences are grouped in the same cluster if these conditions are true:
a minimum of 40 base pair match
greater than 94% identity in the overlap region
a maximum unmatched overhang of 30 base pairs
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Indices: The TIGR database U&T db
TIGR procedure:
Each cluster is assembled using CAP3 assembling program to produce tentative
consensus (TC) sequences.
CAP3 can generate multiple consensus sequences for each cluster
CAP3 rejects chimeric, low-quality and non-overlapping sequences
New TCs resulting from the joining or splitting of previous TCs, get a new TC ID
Build TCs are loaded in the TIGR Gene Indices database and annotated using
information from GenBank and/or protein homology.
Track of the old TC IDs is maintained through a relational database.
References:
Quackenbush et al. (2000) Nucleic Acid Research,28, 141-145.
Quackenbush et al. (2001) Nucleic Acid Research,29, 159-164.
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EST clustering pipeline summary
Unigene
TIGR
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In house databases summary
trEST
trEST is an attempt to produce contigs from UniGene clusters and to translate them into proteins.
This is a two-step process:
-assembly of contigs from a collection of ESTs
-translation of the assembled contigs into protein
Hence, it must be stressed that trEST entries are NOT real protein sequences. They are
hypothetical and are known to contain errors. These data are provided because they might help
biologists to find which UniGene cluster(s) may be relevant for their work.
Unigene
TIGR
In house
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Blast searching EST databases PE
BLAST search against EST databases with a genomic C. Elegans sequence
Introns
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Blast searching EST databases PE
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c
D
N
A
3
5
3
5
Blast searching EST databases PE
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Blast searching EST databases PE
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Blast searching EST databases PE
BLAST search against EST databases with a C. Elegans sequence
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Blast searching EST databases PE
Same clone
Sequenced on the reverse strand
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Contact
with the
authors
Blast searching EST databases PE
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Blast searching EST databases PE
EST assembly to reconstruct a complete sequence
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EST5'.+ CGANGGCCTATCAACAATGAAAGGTCGAAACCTGCGTTTACTCCGGATACAAGATCCACC

EST5'.+ CAGGACACGGNAAAGAGACTTGTCCGTACTGACGGAAAGGTCCAAATCTTCCTCAGTGGA
EST5'.+ AAGGCACTCAAGGGAGCCAAGCTTCGCCGTAACCCACGTGACATCAGATGGACTGTCCTC

EST5'.+ TACAGAATCAAGAACAAGAAGGGAACCCACGGACAAGAGCAAGTCACCAGAAAGAAGACC
EST3'.- AAGAGCAAGTCACCAGAAAGAAGACC
EST5'.+ AAGAAGTCCGTCCAGGTTGTTAACCGCGCCGTCGCTGGACTTTCCCTTGATGCTATCCTT
EST3'.- AAGAAGTCCGTCCAGGTTGTTAACCGCGCCGTCGCTGGACTTTCCCTTGATGCTATCCTT

EST5'.+ GCCAAGAGAAACCAGACCGAAGACTTCCGTCGCCAACAGCGTGAACAAGCCGCTAAGATC
EST3'.- GCCAAGAGAAACCAGACCGAAGACTTCCGTCGCCAACAGCGTGAACAAGCCGCTAAGATC
EST5'.+ GCCAAGGATGCCAACAA
EST3'.- GCCAAGGATGCCAANAAGGCTGTCCGTGCCGCCAAGGCTGCTNCCAACAAGGNAAAGAAG
EST3'.- GCCTCTCAGCCAAAGACCCAGCAAAAGACCGCCAAGAATNTNAAGACTGCTGCTCCNCGT

EST3'.- GTCGGNGGAAANCGATAAACGTTCTCGGNCCCGTTATTGTAATAAATTTTGTTGACC
Blast searching EST databases PE
EST assembly to reconstruct a complete sequence
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Blast searching EST databases PE
EST assembly to reconstruct a complete sequence
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EST1.+ GTTTAATTACCCAAGTTTGAGATTCGTCAAGCGAGGGCCTATCAACAATGAA-GGTCGAA
EST5'.+ CGANGGCCTATCAACAATGAAAGGTCGAA
EST1.+ ACCTGCGTTTACTCCGGATACAAGATCCACCCAGGACACGG-AAAGAGACTTGTCCGTAC
EST5'.+ ACCTGCGTTTACTCCGGATACAAGATCCACCCAGGACACGGnAAAGAGACTTGTCCGTAC
EST1.+ TGACGGAAAGGTCCAAATCTTCCTCAGTGGAAAGGCACTCAAGGGAGCCAAGCTTCGCCG
EST5'.+ TGACGGAAAGGTCCAAATCTTCCTCAGTGGAAAGGCACTCAAGGGAGCCAAGCTTCGCCG
EST1.+ TAACCCACGTGACATCAGATGGACTGTCCTCTACAGAATCAAGAACAAGAAGGGAACCCA
EST5'.+ TAACCCACGTGACATCAGATGGACTGTCCTCTACAGAATCAAGAACAAGAAGGGAACCCA
EST1.+ CGGACAAGAGCAAGTCACCAGAAAGAAGACCAAGAAGTCCGTCCAGGTTGTTAACCGCGC
EST5'.+ CGGACAAGAGCAAGTCACCAGAAAGAAGACCAAGAAGTCCGTCCAGGTTGTTAACCGCGC
EST3'.- AAGAGCAAGTCACCAGAAAGAAGACCAAGAAGTCCGTCCAGGTTGTTAACCGCGC
EST1.+ CGTCGCTGGACTTTCCCTTGATGCTATCCTTGCCAAGAGAAACCAGACCGAAGACTTCCG
EST5'.+ CGTCGCTGGACTTTCCCTTGATGCTATCCTTGCCAAGAGAAACCAGACCGAAGACTTCCG
EST3'.- CGTCGCTGGACTTTCCCTTGATGCTATCCTTGCCAAGAGAAACCAGACCGAAGACTTCCG
EST1.+ TCGCCAACAGCGTGAACAAGCCGCTAAGATCGCCAAGGATGCCAACAAGGCTGTCCGTGC
EST5'.+ TCGCCAACAGCGTGAACAAGCCGCTAAGATCGCCAAGGATGCCAACAA
EST3'.- TCGCCAACAGCGTGAACAAGCCGCTAAGATCGCCAAGGATGCCAAnAAGGCTGTCCGTGC
EST1.+ CGCCAAGGCTGCTGCCAACAAGGAAAAGAAGGCCTCTCAGCCAAAGACCCAGCAAAAGAC
EST3'.- CGCCAAGGCTGCTNCCAACAAGGNAAAGAAGGCCTCTCAGCCAAAGACCCAGCAAAAGAC
EST1.+ CGCCAAGAATGTGAAGACTGCTGCTCCACGTGTCGGAGGAAAGCGATTAAACGTTCTCGG
EST3'.- CGCCAAGAATN TNAAGACTGCTGCTCCNCGTGTCGGNGGAAANCGA-TAAACGTTCTCGG
Blast searching EST databases PE
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CONTIG --------------------------------------------------------------------------------------CGANGGCCTATCAACAATGAAAGGTCGAAACCTG
Genomic AGCTACAAACAGATCCTTGATAATTGTCGTTGATTTTACTTTATCCTAAATTTATCTCAAAAATGTTGAAATTCAGATTCGTCAAGCGAGGGCCTATCAACAATG-AAGGTCGAAACCTG
*** ************ ** * **************

CONTIG CGTTTACTCCGGATACAAGATCCACCCAGGACACGGNAAAGAGACTTGTCCGTACTGACGGAAAG-------------------------------------------------------
Genomic CGTTTACTCCGGATACAAGATCCACCCAGGACACGG-AAAGAGACTTGTCCGTACTGACGGAAAGGTGAGTTCAGTTTCTCTTTGAAAGGCGTTAGCATGCTGTTAGAGCTCGTAAGGTA
************************************ ****************************

CONTIG ------------------------------------------------------------------------------------------------------------------------
Genomic TATTGTAATTTTACGAGTGTTGAAGTATTGCAAAAGTAAAGCATAATCACCTTATGTATGTGTTGGTGCTATATCTTCTAGTTTTTAGAAGTTATACCATCGTTAAGCATGCCACGTGTT
CONTIG ----------------------------------------------GTCCAAATCTTCCTCAGTGGAAAGGCACTCAAGGGAGCCAAGCTTCGCCGTAACCCACGTGACATCAGATGGAC
Genomic GAGTGCGACAAACTACCGTTTCATGATTTATTTATTCAAATTTCAGGTCCAAATCTTCCTCAGTGGAAAGGCACTCAAGGGAGCCAAGCTTCGCCGTAACCCACGTGACATCAGATGGAC
**************************************************************************
CONTIG TGTCCTCTACAGAATCAAGAACAAGAAG---------------------------------------------GGAACCCACGGACAAGAGCAAGTCACCAGAAAGAAGACCAAGAAGTC
Genomic TGTCCTCTACAGAATCAAGAACAAGAAGGTACTTGAGATCCTTAAACGCAGTTGAAAATTGGTAATTTTACAGGGAACCCACGGACAAGAGCAAGTCACCAGAAAGAAGACCAAGAAGTC
**************************** ***********************************************
CONTIG CGTCCAGGTTGTTAACCGCGCCGTCGCTGGACTTTCCCTTGATGCTATCCTTGCCAAGAGAAACCAGACCGAAGACTTCCGTCGCCAACAGCGTGAACAAGCCGCTAAGATCGCCAAGGA
Genomic CGTCCAGGTTGTTAACCGCGCCGTCGCTGGACTTTCCCTTGATGCTATCCTTGCCAAGAGAAACCAGACCGAAGACTTCCGTCGCCAACAGCGTGAACAAGCCGCTAAGATCGCCAAGGA
************************************************************************************************************************
CONTIG TGCCAACAAGGCTGTCCGTGCCGCCAAGGCTGCTNCCAACAAG-----------------------------------------------------------------------------
Genomic TGCCAACAAGGCTGTCCGTGCCGCCAAGGCTGCTGCCAACAAGGTAAACTTTCTACAATATTTATTATAAACTTTAGCATGCTGTTAGAGCTTGTAAGGTATATGTGATTTTACGAGTGT
********************************** ********

CONTIG -------------------------------------------------------------------------------------------------------------------GNAAA
Genomic GTTATTTGAAGCTGTAATATCAATAAGCATGTCTCGTGTGAAGTCCGACAATTTACCATATGCATGAAATTTAAAAACAAGTTAATTTTGTCAATTCTTTATCATTGGTTTTCAGGAAAA
* ***
CONTIG GAAGGCCTCTCAGCCAAAGACCCAGCAAAAGACCGCCAAGAATNTNAAGACTGCTGCTCCNCGTGTCGGNGGAAANCGATAAACGTTCTCGGNCCCGTTATTGTAATAAATTTTGTTGAC
Genomic GAAGGCCTCTCAGCCAAAGACCCAGCAAAAGACCGCCAAGAATGTGAAGACTGCTGCTCCACGTGTCGGAGGAAAGCGATAAACGTTCTCGGTCCCGTTATTGTAATAAATTTTGTTGAC
******************************************* * ************** ******** ***** **** * *********** ***************************

CONTIG C-----------------------------------------------------------------------------------------------------------------------
Genomic CGTTAAAGTTTTAATGCAAGACATCCAACAAGAAAAGTATTCTCAAATTATTATTTTAACAGAACTATCCGAATCTGTTCATTTGAGTTTGTTTAGAATGAGGACTCTTCGAATAGCCCA
*
exon
exon
exon
exon
e
x
o
n
intron
intron
intron
Blast searching EST databases PE
Alignment of an EST contig and a genomic sequence
VI, 2004
Page 52
ORESTES
Goal: to obtain EST sequences from the under
represented, often coding, central portions of mRNAs
Methodology: use low-stringency semi-random priming
followed by PCR, producing low complexity libraries
Results: over 1000000 ESTs produced, of which half
produce novel information
Concluding remarks conclusion
Cons:
- low quality data
- native databases
- 3 ends are heavily represented
- bad/no annotation
- Gene Indices
- (see course)
Pros:
- fast & cheap (automated techniques)
- indispensable for gene structure prediction, gene discovery and genome mapping
(large / small scale)
- efforts:
- normalized databases
- good annotation
- improvements (pre-processing, clustering, assembling)
- ORESTES
- Emerging Gene indices (HUGO, ENSEMBL)
Futur of ESTs:
In human and mouse, most will come as byproducts
of full-length projects,
There are good arguments for trying to reach
saturation on selected tissues
ESTs are still the tool of choice for rapid
exploration of the transcriptomes of various species,
especially with large genomes
ESTs could form a very solid basis for evolutionary
studies