New generation sequencing equiplments The bead-amplification sequencing (Roche/454FLX) Sequencing by synthesis (Illumina/Solexa Genomeanalyzer) Sequencing by ligation (Applied Biosystems SOLiDSystem)
PyrosequencingStep 1 A sequencing primer is hybridizedto a single-stranded PCR ampliconThat serves as a template. Mixtures incubated with theenzymesDNA polymerase, ATP sulfurylase,luciferase, and apyrase as well as thesubstrates, adenosine 5 phosphosulfate (APS), andluciferin.
Step 2The dNTP is added to the reaction.DNA polymerase catalyzes theincorporation of that dNTP intothe DNA strand, if it iscomplementary to the base in thetemplate strand and isaccompanied by release ofpyrophosphate (PPi) in aquantity equimolar to the amountof incorporated
Step 3 ATP sulfurylase converts PPi to ATP in the presence ofAPS. ATP drives the luciferase-mediated conversion ofluciferin to oxyluciferin that generates visible lightproportional to the amount of ATP. The light produced is detected by a CCD chip and seenas a peak in the Pyrogram. The height of each peak (light signal) is proportionalto the number of nucleotides incorporated. Apyrase degrades unincorporated nucleotides .
The bead-amplification sequencing(Roche/454FLX) The DNA sample is sheared into small fragments thatare then attached to 26 µm beads, one fragment to onebead. Then, in a process called emulsion PCR, theDNA is amplified so that each bead carries 100,000copies of the original DNA fragment. The DNA coated beads are then loaded into the wellsOf 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-opticmaterial so that they can transmit the light signals viaa CCD chip. The camera records all the wells in which that base wasadded, and the pyrograms are produced Then that base is washed away and the second base isadded, and so on, until the sequence of the fragment isestablished.
Sequencing by ligation (Applied BiosystemsSOLiD System)
Life Technologies SOLiD 3PlusIllumina GenomeAnalyzer IIxRoche GS FLX
Reference Mihai Pop, Steven L. SalzbergBioinformaticschallenges of new sequencing technologyTrends inGenetics, Volume 24, Issue 3, March 2008, Pages 142-149 M. Margulies et al.Genome sequencing inmicrofabricated high-density picolitre reactors.Nature,437 (2005), pp. 376–380 Bentley DR. Balasubramanian S, Swerdlow, HP, et al:Accurate whole human genome sequencing usingreversible terminator chemistry. Nature 2008:456:53–59