Expressed Sequence Tags - Applied Bioinformatics - Notes | BIT 150, Study notes of Bioinformatics

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Lecture 11 - Expressed Sequence Tags (ESTs) • Baxevanis and Ouellette, chapter 12 (2nd edition • http://www.ncbi.nlm.nih.gov/About/primer/est.html Genome Transcriptome Proteome Metabolome Phenome ,ATG CGA CAA TAG 5 Tt ott ott 3 gttttit tit tits, TAC GCT GTT ATC Transcription Initiation Stop codon codon AUG CGA CAA »» UAG UAC GCU GUU PTL Met Ala Glu Translation Met Fy Ala Fy Glu } Non-transcribed DNA strand (sense) } Transcribed DNA strand (anti-sense) } Messenger RNA (mRNA) with codons Transfer RNA (tRNA) with amino acids attached to anti-codons Amino acid sequence of polypeptide as determined by the sequence of } the first nine nucleotides in the anti- sense strand of the gene and preserved through transcription and translation Complementary DNA cDNA Poly-A tail mRNA STEP 1 Isolate single-stranded 3 MMATTITITITIIITI TIT TTT 5 reid messenger RNA STEP 2 Anneal poly-dT primer MMMM TITTTITITTITT ITT TTT TTT © to poly-A tail of MRNA ' ' ' I ' TITTT | Poly-dT primer STEP 2 Add reverse transcriptase 3' AAAAA 5 rriid enzyme to synthesize I ti trand brid complementary strani 5° TTTTT 3 | STEP 4 Remove mRNA strand 5' TTTTT with RNAseH enzyme biti TITTTTTTTTTTTTTTTTTT! TY | . STEP 5 Se eM rene TETITTTITITIITI TTI ITT IT complementary to remaining ' uw I ' DNAstrand using DNA” y AALAAFILILILTILILLILIILIL LL polymerase | | STEP 6 Recombinant Insert double-stranded Plasmi \q Transformation cDNA into plasmid + J vector (see Box 4.1) and transform into E. coli Transformed E. coli cell Competent E. coli cell DNA cloning in bacteria plasmids Plasmid Chromosomal DNA DNA STEP 1 Oo gS Bacterial cell a E. coli cell containing plasmid to be used as vector STEP 2 Plasmid vector removed #4 = EcoRI and cleaved with EcoRI Plasmid DNA STEP 3 Intact foreign 5° ¢ DNA to be 1 cloned ae a STEP 4 Foreign DNA AAT-T-G cleaved with I EcoRI and G fragment isolated from flanking DNA Isolated DNA fragment STEP 5 Foreign DNA fragment 48-8 ligated with plasmid vector using the enzyme DNA ligase * = DNA ligase Recombinant O° lasmid pl ir \ Transformation STEP 6 Recombinant plasmid transformed into E. coli 7 Transformed E. coli cell Competent E. coli cell Fig 2. http://www.ncbi.nlm.nih.gov/About/primer/est.html at mRNA ve Transcriptase a+ RNA a+ cDNA Ribonuclease degredation of RNA Synthesis of Second Strand of DNA Double Stranded DNA Forward Sequencing Reverse Sequencing Primer Primer > S'EST 3' EST  —__ NCBI dbEST SE Se http: //www.ncbi.nim.nih.gov/dbEST/index.html & Q> NCBI dbEST E ds T > vical mpressed Sequence ags tl Entrez BLAST oY Eee Structure SeachESTforts—“i‘s™S™S*;*~:;S modified during the last | 10 Years + } NCBI > What is dbEST? STN dbEST (Nature Genetics 4:332-3;1993) is a division of GenBank that contains sequence data and other information on "single- pass" cDNA sequences, or Expressed Sequence Tags, from a number of organisms. A brief account of the history of human ESTs in GenBank is available (Trends Biochem. Sci. 20:295- 6;1995). Also, consult the special "Genome Directory" issue of Nature (vol. 377, issue 6547S, 28 September 1995). > Other ways to access dbEST Other ways to access dbEST > How to submit data O00 dbEST Summary <> G + | Shttp://www.ncbi.nim.nih.gov/dbEST/dbEST_summary.htm! - Q> NCBI dbEST °} ye dbEST: database of "Expressed 0 eae Sequence Tags" dbEST release 101405 Summary by Organism - October 14, 2005 Number of public entries: 30,014,098 Homo sapiens (human) 6,134,812 Mus musculus + domesticus (mouse) 4,686,083 Xenopus tropicalis 1,038,272 Rattus sp. (rat) 704,494 Bos taurus (cattle) 702,434 Ciona intestinalis 684,319 Danio rerio (zebrafish) 673,076 Zea mays (maize) 604,279 Triticum aestivum (wheat) 600,039 Gallus gallus (chicken) 578,416 Sus scrofa (pig) 502,500 Xenopus laevis (African clawed frog) 473,761 Arabidopsis thaliana (thale cress) 420,789 Oryza sativa (rice) 406,790 Hordeum vulgare + subsp. vulgare (barley) 394,996 Drosophila melanogaster (fruit fly) 383,407 Glycine max (soybean) 355,970 Canis familiaris (dog) 349,306 - Pinus taeda (loblolly pine) 329,469 - Caenorhabditis elegans (nematode) 302,080 ’ a  L<> jie ji + c S NCBI arte gee le tg) ea roar teen fo) eines) Map Record =— dbEST ae S nitp:/ /www.ncbi.nim.nih.gov/dbEST/how_to_submit.ntm| -Q> NCBI dbEST ° dbEST: database of “Expressed 0 Sequence Tags" Entrez BLAST ce) BE ee ti Structure [Submitting sequences to dbEST and GenBank Expressed Sequence Tags (ESTs) are short (usually about 300-500 bp), single-pass sequence reads from MRNA (cDNA). Typically they are produced in large batches. They represent a snapshot of genes expressed in a given tissue and/or at a given developmental stage. They are tags (some coding, others not) of expression for a given cDNA library. Additional information about ESTs can be found in: i MS, Low T 2v CM. 1993. dbEST--database for "expressed sequence tags." Nat Genet 4(4):332-333. Most EST projects develop large numbers of sequences. These are commonly submitted to GenBank and dbEST as batches of dozens to thousands of entries, with a great deal of redundancy in the citation, submitter and library information. To improve the efficiency of the submission process for this type of data, we have designed a special streamlined submission process and data format. dbEST also includes sequences that are longer than the traditional ESTs, or are produced as single sequences or in smal] batches. Among these sequences are products of differential display experiments and RACE experiments. The thing that these sequences have in common with waditional ESTs, regardless of length, quality, or quantity, is that there is little information that can be annotated in the record. If a sequence is later characterized and annotated with biological features such as a coding region, 5'UTR, or 3'UTR, it should be submitted through the regular GenBank submissions procedure (via Banklt or Sequin), even if part of the sequence ae  Q> NCBI dbEST UniGene ORGANIZED VIEW OF THE TRANSCPUPTOME y <> NCBI Pood ced [Limits | Previewlindex | History | Clipboard | Details | UniGene is an experimental system for automatically partitoning GenBank sequences into 2 non- recundant set of gene-oriented clusters. Each UniGene cluster contains sequences that represent @ unique gene, as well as related information such as the tissue types in which the gene has been expressed and map location. Dee Species UniGene Entnes er eal) Chordata Mammalia Bos taurus 39,048 Canis familiaris 22,930 Homo sapiens 54,576 Macaca mulatta 4,701 er ee ea Mus musculus 43,104 Ovis aries 1,714 Rattus norvegicus 38,675 Sus scrofa 32,069 Reet aia Aves _ Gallus gallus 30,221 HomoloGene Amphibia 7 Xenopus laevis 28,040 ead Xenopus tropicalis 33,132 Actinopterygii nhac Danio pois 31,681 = Fundulus heteroclitus 3,154 td Oncorhynchus mykiss 24,362 Oryzias latipes 9,848 Sacaaald Salmo salar 8,371 Takifugu rubripes 2,355 MGC cDNA clones Ascidiacea Ciona intestinalis 14,373 ares LN Ciona savignyi 6,315 Projects Molgula tectiformis 7,351 fe) OO UniGene =| <> Cc + S http: //www.ncbi.nim.nih.gov/UniGene/UGOrg.cgi?TAXID=3352 = Q> NCBI dbEST ee PubMed Protein Genome Structure PopSet Taxonomy OMIM Search UniGene ~3) Pinus taedaforganism| (Go) (Clear ) Limits Index History Details Pinus taeda: UniGene Build #21 Lineage:cellular organisms; Eukaryota; Viridiplantae; Streptophyta; Charophyta/Embryophyta group; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Coniferophyta; Coniferopsida; Coniferales; Pinaceae; Pinus; Pinus; Pinus taeda Known genes are from GenBank 06 Sep 2005 ESTs are from dbEST through 06 Sep 2005 UniGene Links 108 mRNAs Library Browser 0 Models DDD 0 HTC Q Ti 62,100 EST, 3'reads ery Nps 140,375 EST, S'reads FAQ 27,037 EST, other/unknown 229,620 total sequences in clusters Other Links uild M Transcript Base Gene Alignments between all transcript sequences are used to generate HomoloGene clusters of sequences originating from the same gene. More... dbEST Trace Archive N sters (« BLAST 14,198 sets total CGAP 89 sets contain at least one MRNA 0 sets contain at least one HTC sequence 14,188 sets contain at least one EST 79 sets contain both mRNAs and ESTs  <a. The Institute for Genomic Research -- Gene Indices Cl) + |) Si hup:/swww.tigr.org/tdb/tgi/plant.shumt 7 Qy NCBI dbEST 44 TIGR The Insituiesror’ Ge th is we Home Research Groupe | Scientitic Progrems © Database Home Gene Indices © Lomerechonsivo Microbial Resources What's New | Blast | TG1 Software | £60 | Genomic Maps | Resourcer | FAQ hk Unfinished Microbia ® Genomes © Plant Genomics © Parasites Databases Per) Fead ult eet) PEE] a 4.0. ee andy ; Pe 1 de © Fungal Databa: © [brelvtic Ruminel Bacteria ee Pel Bre) VEE sites butler ay [ETzs Genome Properties Database TGI Data 5 amy “a ee