SOLEXA aka Sequencing by Synthesis 1 Randomly cut

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 ss. DNA templates. After each round of amplification, the strands are denatured to create more templates.

3) Add four labeled reversible terminators, primers, and DNA polymerase. 4) Wash off all of the unbound components, excite the laser, and read the emitted fluorescence of the first base for each cluster. 5) The blocked 3’ terminus and fluorophore has to be removed before repeating to determine the next base

454 Sequencing • Capable of sequencing 400 -600 megabases of DNA in a 10 hour run • Applications: 1. Whole genome sequencing 2. Amplicon sequencing 3. Transcriptome sequencing 4. Metagenomics • Advantages: low cost, longer reads and higher accuracy than the Sanger chaintermination method

454 Sequencing Concept • Large-scale parallel pyrosequencing, “sequencing by synthesis”: 1. Each nucleotide is added in turn, producing PPi 2. Sulfurylase uses PPi to produce ATP, that is used by luciferase to produce light 3. Apyrase denatures the remaining d. NTP at each step 4. The light produced with the addition of each nucleotide can be plotted to form a sequence

$454 Sequencing Technology • DNA is fractionated, 5’ biotin labeled and attached to streptavidin$

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