Transcript Expressed sequence Tags (EST)

Expressed
sequence
Tags (EST)
Aurora Kraus MD*, JD*, PhD*,
*Pending
Expressed sequence tag
• Expressed
• Sequence
• Tags
Importance
• Gene exploration
• Populating databases
• Random
• Cheap + quick(er) at time
• Less interested in accuracy
• Functional – phenotype related
• mRNA sequenced
• Distilled information
De, I., Junqueira-De-Azevedo, L. M. & Ho, P. L. A
survey of gene expression and diversity in the
venom glands of the pitviper snake Bothrops
insularis through the generation of expressed
sequence tags (ESTs). Gene 299, 279–291 (2002).
History
• 1980s -- sequencing new and expensive
• ESTs are small – 400-600bp
• No databases
• No idea of what genetic material does what yet
• Craig Venter
Populating Databases
https://journals.tdl.org/jodi/index.php/jodi/article/view/5878/5882
Craig Venter
J. Craig Venter. Et al. rapid cDNA
sequencing (EST) directionally clones
human infant brain cDNA library. Nat.
Genet. 4, 373–380 (1993).
Adams, Mark D.. Kerlavage, Anthony R..
Fields, C. J. C. V. 3400 new expressed
sequence tags identify diversity of
transcripts in human brain. Nat. Genet. 4,
256–267 (1993).
Organism/tissue
cDNA library
generation:
Cloning
mRNA
cDNA
Random
sequencing
Anatomy of mRNA
• EST Goal: to sequence
pieces of the coding region
mRNA – learn functional
information
• Directional cloning
• Oligo(dT) primers
• Bind to poly-A tail
• Result: most ESTs match to
the 3’UTR and some
coding region
• Small size
• Missing info
TTTTTTT
Random Sampling
• Pick random colonies
• Sequence more of most
common RNA  rRNA
1 – RNA without DNA
purification
2 RNA after DNase treatment
1 23
DNA
28s
18s
3 poly-A mRNA purification
 Increase probability
sequence novel mRNA
Walker, J. M. Expressed Sequence tags (ESTs)Generation and analysis.
(2009). doi:doi: 10.1007/978-1-60327-136-3_1
Other optimization
techniques
5’ UTR
• ORE EST
• Targeting the coding
region of mRNA
• Directional cloning:
Primers target poly-A tail =
bias to 3’end
• Gaps!
• SSH
• Excluding
repetitive/abundant rRNA
• Goal: more unique content
Dias Neto, E. et al. Shotgun sequencing of
the human transcriptome with ORF
expressed sequence tags. PNAS 97, 3491–
3496 (2000).
Coding region
3’ UTR
Uses today
• Next Gen Sequencing =
Mammoth amounts data
• ESTs help mine the data
• Mapping ESTs to
RNAseq and
Microarray data
• Relating big data back
to phenotype
• Non-model organism
research
• Lack genomes
• Microarray design
• Phylogenomics
Bibliography
1. De, I., Junqueira-De-Azevedo, L. M. & Ho, P. L. A survey of gene expression and diversity in the venom glands of the pitviper snake Bothrops insularis
through the generation of expressed sequence tags (ESTs). Gene 299, 279–291 (2002).
2. J. Craig Venter. rapid cDNA sequencing (EST) directionally clones human infant brain cDNA library. Nat. Genet. 4, 373–380 (1993).
3. Adams, Mark D.. Kerlavage, Anthony R.. Fields, C. J. C. V. 3400 new expressed sequence tags identify diversity of transcripts in human brain. Nat.
Genet. 4, 256–267 (1993).
4. Walker, J. M. Expressed Sequence tags (ESTs)Generation and analysis. (2009). doi: 10.1007/978-1-60327-136-3_1
5. Bork, P. & Schuler, G. D. Bioinformatics: Bits and Bytes Pieces of the puzzle: expressed sequence tags and the catalog of human genes The Puzzle of
Life. J. Mol Med 75, 694–698 (1997).
6. Hillier, L. et al. Generation and Analysis of 280,000 Human Expressed Sequence Tags. Genome Res. 6, 807–828 (1996).
7. Dias Neto, E. et al. Shotgun sequencing of the human transcriptome with ORF expressed sequence tags. PNAS 97, 3491–3496 (2000).
8. Hardwick, C. Expressed sequence Tags - ESTablishing bridges between genomes. Sci. Mol. Cell. Biol. J. Cell Biol. Sci. Sci. Sci. Sci. Mol. Cell. Biol. Mol. Cell.
Biol.J. Cell Biol. Nat. Cell Proc. Natl. Acad. Sci. U. S. A.J. Cell Biol. J. Cell Biol. Mol. Biol. Cell 273, (1996).
9. Mark Adams, Jenny Kelley, Jeannine Gocane, Micheal Polymeropoulos, Hong Xiao, Anthony Kelley, Richard McCombie, C. V. complementary DNA
sequencing: Expresses sequence tags and the human genome project. Science (80-. ). 252, 1651–1656 (1991).