WWU Genomic Project pilot

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WWU Genomic Projectpilot

• Nasonia vitripennis
• EST Sequencing

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Expressed Sequence TagsESTs

• End sequenced cDNAs.

• cDNA synthetic DNA transcribed from a specific
  mRNA,
• through the action of the enzyme reverse
  transcriptase,

• reverse transcriptase catalyzes the the
  synthesis of a DNA strand from an RNA template.

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Expression Specific LibrarycDNA template source

• mRNA (or total RNA) is extracted,
• mRNA from developmental time points (temporal).
• mRNA from specific tissues (spatial),
• mRNA from tissue under differential conditions,
• environmental (nurture),
• genetic (nature),
• etc.
• mRNA is converted to complementary DNA,
• cDNA is inserted into a vector.

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cDNA Librarysequence template source

• Snapshot of the genes expressed in a given
  tissue at a given time,
• May be manipulated to keep relative transcript
  abundance,
• provides some indication as to what is being
  expressed,
• and how much, in relation to other transcripts
  from the sample.
• May be enriched for low copy (rare) cDNAs,
• May be put into expression vectors, etc.,
• remember, bacteria dont process introns.

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Clontech Smart cDNA Library Construction Kit
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PolyA RNA
First strand synthesis coupled with (dC) tailing
by Reverse Transcriptase...
Template switching and extension by RT...
PCR amplify
SfiI A/B 13 bp cutters
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Continued...
Amplified ds cDNAs
SfiI digest
Ligate into double digested lTriplEx2
Package in host cells
cDNA Library
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EST Genome Projectshigh throughput

• ... cDNAs are randomly sequenced from cDNA
  libraries in order to gain rapid access to the
  genes in a genome,
• vector sequences are used for cycle sequencing
  primers,
• means to identify transcribed genes,
• used in conjunction with Bioinformatics, provides
  putative genes with functional annotation,
• provides resources for genomic comparisons and
  genome mapping,
• facilitates the identification of orthologs and
  paralogs for comparative genomics.

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Expressed Sequence TagsStrategies
3 Sequencing 3 UTR is generally the most
diverged... ...differentiates gene family
members... however, sometimes lacks enough
translated sequence for gene identification.
5 Sequencing 5 end sequencing, facilitates
functional identification,
best EST libraries... cDNAs are manipulated
so that complete DNA sequences are obtained.
...if clones are maintained, other end
sequencing can be performed on interesting cDNAs.

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Best ETSs End sequencing of Digested cDNAs

• ... restriction digests are performed prior to
  cloning the cDNA into the vector,
• large numbers of sequences are obtained,
  overlapping sequences are used to build
  complete cDNA sequences.

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TIGR DatabaseThe Institute for Genome Research

• not-for-profit institute,
• screens, clusters and assembles ESTs in a highly
  rigorous manner,
• separates closely related genes into distinct
  consensus sequences,
• separates splice variants,
• produces long representations of the underlying
  gene sequences.

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dbEST release 050203 Summary by Organism - May
2, 2003 Number of public entries
16,547,527 Homo sapiens (human)
5,094,900 Mus musculus domesticus (mouse)
3,721,428 Rattus sp. (rat)

525,545 Ciona intestinalis
492,488 Gallus
gallus (chicken)
418,093 Triticum aestivum (wheat)
415,747 Hordeum
vulgare subsp. vulgare (barley)
340,945 Bos taurus (cattle)
319,725 Danio rerio
(zebrafish)
311,335 Glycine max (soybean)
308,582 Xenopus laevis
(African clawed frog)
274,975 Drosophila melanogaster (fruit fly)
261,271 Zea mays (maize)

210,748 Oryza sativa (rice)
202,290 Caenorhabditis
elegans (nematode)
192,132 Medicago truncatula (barrel medic)
185,621 Arabidopsis thaliana
(thale cress)
178,538
Silurana tropicalis
165,097 Dictyostelium discoideum
155,032 Lycopersicon esculentum
(tomato) 150,193 Chlamydomonas
reinhardtii 140,457 Sus
scrofa (pig)
131,330 Oryzias latipes (Japanese medaka)
103,098 Oncorhynchus mykiss (rainbow
trout) 101,845 Anopheles gambiae
98,840 Solanum tuberosum (potato)
94,423 Sorghum bicolor (sorghum)
89,619 Vitis vinifera

86,102 Lactuca sativa
68,188 Toxoplasma gondii
63,637 Pinus taeda (loblolly pine)
60,226 Salmo salar

58,330 Populus tremula x Populus tremuloides
56,013 Physcomitrella patens subsp. patens
49,583 Helianthus annuus
46,951 Schistosoma
japonicum (blood fluke)
45,900 Ascaris suum (pig roundworm)
39,242 Gossypium arboreum
38,894 Lotus japonicus

36,210 Bombyx mori (domestic silkworm)
28,969
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Guppy Level
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Realistic Goals
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The Dream
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June 5, 2003 Dear
Researcher, Im sorry to inform you, but we have
now sequenced ESTs from the parasitoid wasp
Nasonia vitripennis. Your genomic project,
sequencing expressed tags from organism of choice
, has recently been
surpassed in scale by undergraduate students at
Western Washington University. etc. etc. etc.
Yours Truly, Carol Trent
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Challenges to Greatness Representation

• PCR amplification favors the most abundant
  templates,
• rare transcripts will be under sampled,
• highly expressed sequences over sampled,
• Technical Difficulties.

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To Do

• Pick/PCR DNA (8 colonies each person),
• teL2 vs. teR2,
• youll streak out the colonies on a fresh, well
  labeled plate.
• Run gel to ID different sized inserts,
• Cycle sequence as many unique 5 ends as is
  possible (up to four per person),
• if less than four unique inserts, we may
  sequence some 3 ends,

teL2
TriplEx2 MCS
SfiA
SfiB
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We will submit good sequences to NCBI with
attribution.