1. SOLEXA aka: Sequencing by Synthesis
1) Randomly cut DNA
fragments and ligate
adaptors to both ends
2) Attach each adaptor to a flow cell
to create ssDNA templates. After
each round of amplification, the
strands are denatured to create
more templates.

2. Add four labeled reversible terminators, primers, and DNA polymerase.
Wash off all of the unbound
components, excite the laser,
and read the emitted
fluorescence of the first base
for each cluster.
The blocked 3’ terminus
and fluorophore has to be
removed before repeating to
determine the next base

3. 454 Sequencing
Capable of sequencing megabases of DNA in a 10 hour run
Applications:
Whole genome sequencing
Amplicon sequencing
Transcriptome sequencing
Metagenomics
Advantages: low cost, longer reads and higher accuracy than the Sanger chain-termination method

4. 454 Sequencing Concept
Large-scale parallel pyrosequencing, “sequencing by synthesis”:
Each nucleotide is added in turn, producing PPi
Sulfurylase uses PPi to produce ATP, that is used by luciferase to produce light
Apyrase denatures the remaining dNTP at each step
The light produced with the addition of each nucleotide can be plotted to form a sequence
Each incorporation releases PPi
Sulfurylase uses PPi to produce ATP, which luciferase can use to generate light
The detection of light by a camera when a new nucleotide is added indicates which nucleotide was next in the sequence
Since ATP is used by luciferase as part of the detection mechanism, dATP-alphaS is used for sequencing (deoxyadenosine alpha-thio triphosphate) that cannot be used by luciferase but still can be incorporated into a sequence like ATP
image:
additional info: (pdf format)

5. 454 Sequencing Technology
DNA is fractionated, 5’ biotin labeled and attached to streptavidin coated beads. The non-labeled strand is released.
The DNA on each bead is amplified onto the bead
One bead is deposited in each well
Pyrosequencing takes place in the Genome Sequencer FLX instrument

6. Applied Biosystems SOLiD System
© Copyright 2008 Applied Biosystems. All Rights Reserved.

7. © Copyright 2008 Applied Biosystems. All Rights Reserved.