Max Bachour

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• Max Bachour

Jessica Chen
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• Shotgun or 454 sequencing

• High throughput sequencing technique that can
  collect a large amount of data at a fast rate.
• Works by partially digesting a genome or big
  strand of DNA into small overlapping fragments
• These small fragments are sequenced and fragments
  that overlap are matched together.

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Steps Behind 454 sequencing

1. The genome is fragmented and the fragments are
   denatured.
2. Fragments are amplified and assigned to beads.
   One fragment per one microbead.
3. Each bead is placed in the wells of a fiber optic
   slide.
4. Packing beads placed in all the wells.

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Steps Behind 454 sequencing

• Solution of one nucleoside is flooded onto tray.
• If base added is next in the sequence, it will be
  added to the single stranded DNA on the bead.
• When a nucleoside is added to DNA, 2 phosphates
  are given out
• Enzymes in packing beads convert phosphate groups
  to ATP and then the ATP to light energy.

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Steps Behind 454 sequencing

• Computer and camera detect light in a certain
  well as a certain base is added to the tray.
• Base is washed off and process is repeated with
  another base.
• End product is large amount of fragments
  sequenced.

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Genome Sequence Analysis

• ?Contig Assembly
• ?Identifying open reading frames (ORF) using gene
  prediction programs

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What is the initial problem with assembly?
Sequenced fragmented DNA
CONTIG 1
CONTIG 2
Incorrectly Assembled DNA Sequence
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How is this problem solved?
Sequenced fragmented DNA
Masked DNA Sequence
Assembled DNA Sequence
CONTIG 3
CONTIG 1
CONTIG 5
CONTIG 4
CONTIG 2
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How do we identify genes?

• Use gene prediction programs (Fgenesh, Genscan,
  Genemark) to determine potential genes also
  determine any repeat sequences
• Enter contig
• Which of the predicted genes are most likely
  existing genes?
• ? Use BLAST

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How do we use BLAST?

• ? tblastn all predicted genes against an EST
  database (ESTDB)
• Why ESTDB? record of all known/identified mRNA
  (cDNA library)
• Why tblastn? -- amino acid sequence more likely
  to be conserved
• ? use blastn and blastp
• -blastp determine expression of gene

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Analyzing BLAST data
Gene 1
Protein sequence MFVVQYLGSSRSWTSCSHSSKPGVDSRGRAEPHLAVGRSSLLGRVQTGLKGGGMKDSDLT
GDSSLARANQSMGICKSEGTVDRRLKSQVSQLLLGLLLIRLEGLLATCMTGPHGDAGAGS
THK

gtgbFC457105.1 UCRVU04_CCNI646_g1 Cowpea 524B Mixed Tissue and Conditions cDNA
Library UCRVU04-1 Vigna unguiculata cDNA clone CCNI646, mRNA
sequence.
Length807

Score 215 bits (548), Expect(2) 2e-55, Method Compositional matrix adjust.
Identities 110/112 (98), Positives 110/112 (98), Gaps 0/112 (0)
Frame -1

Query 12 SWTSCSHSSKPGVDSRGRAEPHLAVGRSSLLGRVQTGLKGGGMKDSDLTGDSSLARANQS 71
SWTSCSHS KPGVDSRGRAEPHLAVGRSSLLGRVQTGLKGGGMKDSDLTGDSSLARANQS
Sbjct 438 SWTSCSHSKPGVDSRGRAEPHLAVGRSSLLGRVQTGLKGGGMKDSDLTGDSSLARANQS 259

Query 72 MGICKSEGTVDRRLKSQVSQLLLGLLLIRLEGLLATCMTGPHGDAGAGSTHK 123
MGICK EGTVDRRLKSQVSQLLLGLLLIRLEGLLATCMTGPHGDAGAGSTHK
Sbjct 258 MGICKEGTVDRRLKSQVSQLLLGLLLIRLEGLLATCMTGPHGDAGAGSTHK 103

• Critical data
• e-value
• match
• EST source

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Advantages and Disadvantages

• Fast sequencing at a high volume
• Cheap compared to other methods
• Much higher coverage protection
• Repetitive sequences can disrupt computer program
  into thinking that unrelated sequences are in
  fact connected.
• More prone to error and missing sequences

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Drastically changed genomics in a very short
amount of time