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.
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
• 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.
• The B adapter contains a 5’ biotin tag used
for mobilization.
• The beads are magnetized and attract the
biotin in the B adaptors.
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
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.
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.
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.
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
• 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.
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QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
PYROSEQUENCING
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.
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.
The Titanium model
• Read lengths of 400-600 base pairs.
• 400-600 million base pairs read per run.
• About 100 million parallel reads
Additional Links
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454 life sciences:
• www.454.com
Detailed overview of the system:
• http://www.454.com/products-solutions/multimediapresentations.asp
Pyrosequencing animation:
• http://www.youtube.com/watch?v=bFNjxKHP8Jc&feature=rel
ated
• http://www.pyrosequencing.com/DynPage.aspx?id=7454
Sequencing step animation:
• http://www.youtube.com/watch?v=kYAGFrbGl6E