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

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

Genome Sequencing Utilizing

Light-Emitting Luciferase and

PCR-Reaction-Mixture-in-Oil Emulsion.

Mr Meir Shachar

Dr Edwin Ginés-Candelaria

• 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

PYROSEQUENCING

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

• 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