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Jessica Chen. Max Bachour. 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

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Presentation Transcript
max bachour
Jessica Chen

Max Bachour

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.

Shotgun or 454 sequencing

steps behind 454 sequencing
Steps Behind 454 sequencing
  • The genome is fragmented and the fragments are denatured.
  • Fragments are amplified and assigned to beads. One fragment per one microbead.
  • Each bead is placed in the wells of a fiber optic slide.
  • Packing beads placed in all the wells.
steps behind 454 sequencing1
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.
steps behind 454 sequencing2
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.
genome sequence analysis
Genome Sequence Analysis

Contig Assembly

Identifying open reading frames (ORF) using gene prediction programs

slide7
What is the initial problem with assembly?

Sequenced fragmented DNA

CONTIG 2

CONTIG 1

Incorrectly Assembled DNA Sequence

slide8
How is this problem solved?

Sequenced fragmented DNA

Masked DNA Sequence

Assembled DNA Sequence

CONTIG 3

CONTIG 1

CONTIG 5

CONTIG 4

CONTIG 2

how do we identify genes
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

how do we use blast
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

analyzing blast data
Analyzing BLAST data
  • Critical data:
    • e-value
    • %match
    • EST source
advantages and disadvantages
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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