PYROSEQUENCING

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

Mr. Meir Shachar Dr. Edwin Ginés-Candelaria

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

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.

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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

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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 aTIFF (LZW) decompressor

are needed to see this picture.

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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

• 454 life sciences:• www.454.com

• Detailed overview of the system:• http://www.454.com/products-solutions/multimedia-

presentations.asp• Pyrosequencing animation:

• http://www.youtube.com/watch?v=bFNjxKHP8Jc&feature=related• http://www.pyrosequencing.com/DynPage.aspx?id=7454

• Sequencing step animation:• http://www.youtube.com/watch?v=kYAGFrbGl6E