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PYROSEQUENCING

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PYROSEQUENCING Genome Sequencing Utilizing Light-Emitting Luciferase and PCR-Reaction-Mixture-in-Oil Emulsion. Mr. Meir Shachar Dr. Edwin Gin s-Candelaria – PowerPoint PPT presentation

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Title: PYROSEQUENCING


1
PYROSEQUENCING
  • Genome Sequencing Utilizing Light-Emitting
    Luciferase and PCR-Reaction-Mixture-in-Oil
    Emulsion.

Mr. Meir Shachar Dr. Edwin Ginés-Candelaria
2
Introduction
  • Read lengths are around 200-300 bases.
  • 400,000 reads of parallel sequencing
  • 100mb of output per run
  • Run time 7.5 hours

Unless otherwise stated, read and output data
are provided on the 454 FLX 20 sequencer
3
Step 1 Preparation of the DNA
  • DNA is fragmented by nebulization
  • The DNA strands ends are made blunt with
    appropriate enzymes
  • A and B adapters are ligated to the blunt
    ends using DNA ligase
  • The strands are denatured using sodium hydroxide
    to release the ssDNA template library (sstDNA).

4
The Adapters
  • The A and B adapters are used as priming sites
    for both amplification and sequencing since their
    composition is known.
  • The B adapter contains a 5 biotin tag used for
    mobilization.
  • The beads are magnetized and attract the biotin
    in the B adaptors.

5
Filtering the Mess
  • There are four adaptor combinations that are
    formed from the ligation.
  • A---sequence---A
  • A---sequence---B
  • B---sequence---A
  • B---sequence---B

6
Step 2 Cloning of the DNA (emPCR)
  • Using water-in-oil emulsion, each ssDNA in the
    library is hybridized onto a primer coated bead.
  • By limiting dilution, an environment is created
    that allows each emulsion bead to have only one
    ssDNA.
  • Each bead is then captured in a its own emulsion
    micro-reactor, containing in it all the
    ingredients needed for a PCR reaction.
  • PCR takes place in each of these beads
    individually, but all in parallel.
  • This activity as a whole is emPCR.

7
Post emPCR
  • The micro-reactors are broken, and the beads are
    released.
  • Enrichment beads are added (containing biotin)
    these attach to DNA rich beads only.
  • A magnetic field filters all DNA rich beads from
    empty beads, and then extracts the biotin beads
    from the DNA rich beads.
  • The DNA in the beads are denatured again using
    sodium hydroxide, creating ssDNA rich beads ready
    for sequencing.

8
Step 3 Sequencing
  • Utilizing the A adapter, a primer is added to the
    ssDNA.
  • The beads are now loaded into individual wells
    created from finely packed and cut fiber-optics
    (PicoTiterPlate device).
  • The size of the wells do not allow more than one
    ssDNA bead to be loaded into a well.
  • Enzyme beads and packing beads are added. Enzyme
    beads containing sulfurase and luciferase, and
    packing beads used only to keep the DNA beads in
    place.
  • Above the wells is a flow channel, passing
    nucleotides and apyrase in a timed schedule.

9
PYROSEQUENCING
10
The Chemical Chain
  • The nucleotide bases are added in a timed fashion
    (beginning with A, T, G, C with 10s between each
    nucleotide and a successive apyrase wash,
    followed by the next nucleotide.)
  • As a bi-product of incorporation, DNA polymerase
    releases a pyrophosphate molecule (PPi).
  • The sulfurylase enzyme converts the PPi into ATP

11
PYROSEQUENCING
12
The Fireworks Show
  • Each ATP produced by sulfurilase is used by
    luciferase.
  • Luciferase hydrolyzes each ATP molecule to
    produce oxy-luciferin and light from the
    substrate luciferin.
  • Luciferin ATP O2 ?(luciferase)?
  • AMP oxy-luciferin PPi CO2 light
  • A CCD camera records the light from the reaction.
  • A wash of apyrase is released after each
    nucleotide to remove the unincorporated
    nucleotides.

13
PYROSEQUENCING
14
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15
PYROSEQUENCING
16
Step 4 Data analysis
  • The intensity of the light emitted by luciferase
    is proportional to the number of nucleotides
    incorporated.
  • Therefore, if the intensity of a single read is 3
    times the intensity of a previous read, there are
    3 times the amount of incorporated nucleotides in
    the second read.

17
Two Types of Analysis
  • Run Time Analysis
  • Image acquisition raw image
  • Image processing mapping of raw image to
    corresponding wells
  • Signal processing the individual well signals
    incorporated into a flowgram
  • Post-run Processing (separate computer)
  • Assembly overlaps multiple reads to create
    larger reads assembling a consensus read.
  • Mapping maps the reads onto the consensus
    obtained from the assembly to re-sequence the
    genome.
  • Amplicon Variant Analysis compares the sample
    reads to referenced known sequences for
    identification.

18
The Titanium model
  • Read lengths of 400-600 base pairs.
  • 400-600 million base pairs read per run.
  • About 100 million parallel reads

19
Additional Links
  • 454 life sciences
  • www.454.com
  • Detailed overview of the system
  • http//www.454.com/products-solutions/multimedia-p
    resentations.asp
  • Pyrosequencing animation
  • http//www.youtube.com/watch?vbFNjxKHP8Jcfeature
    related
  • http//www.pyrosequencing.com/DynPage.aspx?id7454
  • Sequencing step animation
  • http//www.youtube.com/watch?vkYAGFrbGl6E
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