The a and b adapters are used as priming sites for both amplification

PYROSEQUENCING
Genome Sequencing Utilizing
Light-Emitting Luciferase and
PCR-Reaction-Mixture-in-Oil
Emulsion.
Mr. Meir Shachar
Dr. Edwin Ginés-Candelaria
Introduction*
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Read lengths are around 200-300 bases.
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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
Step 1: Preparation of
the DNA
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DNA is fragmented by nebulization
•
The DNA strand’s 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).
The Adapters
•
The A and B adapters are used as priming
sites for both amplification and sequencing
since their composition is known.
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The B adapter contains a 5’ biotin tag used
for mobilization.
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The beads are magnetized and attract the
biotin in the B adaptors.
Filtering the Mess
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There are four adaptor combinations that
are formed from the ligation.
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A sequence A
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A sequence B
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B sequence A
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B sequence B
Step 2: Cloning of the
DNA (emPCR)
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Using water-in-oil emulsion, each ssDNA in
the library is hybridized onto a primer
coated bead.

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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.
Post emPCR
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The micro-reactors are broken, and the
beads are released.
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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.
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.
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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.
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Above the wells is a flow channel, passing
nucleotides and apyrase in a timed
schedule.
PYROSEQUENCING
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
PYROSEQUENCING
The Fireworks Show
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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 + O
2
(luciferase)
AMP + oxy-luciferin + PPi + CO
2
+ light
•
A CCD camera records the light from the
reaction.
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A wash of apyrase is released after each
nucleotide to remove the unincorporated
nucleotides.
PYROSEQUENCING
QuickTime™ and a

TIFF (LZW) decompressor
are needed to see this picture.
PYROSEQUENCING
Step 4: Data analysis
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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.
Two Types of Analysis
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Run Time Analysis:
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Image acquisition – raw image
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Image processing – mapping of raw
image to corresponding wells
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Signal processing – the individual well
signals incorporated into a flowgram
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Post-run Processing (separate computer):
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Assembly – overlaps multiple reads to
create larger reads; assembling a

consensus read.
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Mapping – maps the reads onto the
consensus obtained from the assembly
to “re-sequence” the genome.
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Amplicon Variant Analysis – compares
the sample reads to referenced known
sequences for identification.
The Titanium model
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Read lengths of 400-600 base pairs.
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400-600 million base pairs read per run.
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About 100 million parallel reads
Additional Links
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454 life sciences:
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www.454.com
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Detailed overview of the system:
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/>presentations.asp
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Pyrosequencing animation:
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•

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Sequencing step animation:
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