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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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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


Max bachour

What is the initial problem with assembly?

Sequenced fragmented DNA

CONTIG 2

CONTIG 1

Incorrectly Assembled DNA Sequence


Max bachour

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


Max bachour

Drastically changed genomics in a very short amount of time


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