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KALAIVANI.P

BTB-10-014

New generation sequencing equiplments

The bead-amplification sequencing (Roche/454FLX)

Sequencing by synthesis (Illumina/Solexa Genome analyzer)

Sequencing by ligation (Applied Biosystems SOLiDSystem)

PyrosequencingStep 1 A sequencing primer is hybridized

to a single-stranded PCR amplicon

That serves as a template.

Mixtures incubated with the

enzymesDNA polymerase, ATP sulfurylase,

luciferase, and apyrase as well as the

substrates, adenosine 5' phosphosulfate (APS), and luciferin.

Step 2

The dNTP is added to the reaction. DNA polymerase catalyzes the incorporation of that dNTP into the DNA strand, if it is complementary to the base in the template strand and is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated

Step 3 ATP sulfurylase converts PPi to ATP in the presence of

APS.

ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light proportional to the amount of ATP.

The light produced is detected by a CCD chip and seen as a peak in the Pyrogram.

The height of each peak (light signal) is proportional to the number of nucleotides incorporated.

Apyrase degrades unincorporated nucleotides .

Step 3

The bead-amplification sequencing (Roche/454FLX)

The DNA sample is sheared into small fragments that are then attached to 26 µm beads, one fragment to one bead. Then, in a process called emulsion PCR, the DNA is amplified so that each bead carries 100,000 copies of the original DNA fragment.

The DNA coated beads are then loaded into the wells Of a 1.6 million-well picotiter plate so that, on average, there is one bead per well.

The wells of the picotiter plate are made of fiber-optic material so that they can transmit the light signals via a CCD chip.

The camera records all the wells in which that base was added, and the pyrograms are produced

Then that base is washed away and the second base is added, and so on, until the sequence of the fragment is established.

Emulsion PCR

Illumina/Solexa Genome analyzer

Sequencing by ligation (Applied BiosystemsSOLiD System)

Life Technologies SOLiD 3Plus

Illumina Genome Analyzer IIx

Roche GS FLX

Reference Mihai Pop, Steven L. SalzbergBioinformatics

challenges of new sequencing technologyTrends in Genetics, Volume 24, Issue 3, March 2008, Pages 142-149

M. Margulies et al.Genome sequencing in microfabricated high-density picolitre reactors.Nature, 437 (2005), pp. 376–380

Bentley DR. Balasubramanian S, Swerdlow, HP, et al: Accurate whole human genome sequencing using reversible terminator chemistry. Nature 2008:456:53–59

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