1. High-throughput sequencing techniques
complex DNA sample
adapter ligation
PCR amplification & sequencing

2. 1) Prepare Adapter Ligated ssDNA Library
454 Process Overview
1) Prepare Adapter Ligated ssDNA Library
2) Clonal Amplification
on 28 µ beads
3) Load beads and enzymes
in PicoTiter Plate™
4) Perform Sequencing by synthesis
on the 454 Instrument of
~250,000 molecules

3. 454 Sequencing

4. 454 base calling
Key sequence = TCAG for identifying wells and calibration
Flow Order
TACG

6. Titration of beads to DNA template
After emulsion
PCR
Beads separated for pyrosequencing

7. Titration of beads to DNA template
After emulsion
PCR
Beads separated for pyrosequencing

8. Limitations of Roche/454 Homopolymers Out-of-phase
One bead/bubble deviations
No simple paired-end sequencing

9. Illumina Library Contruction

10. Solexa/Illumina

11. Solexa / Illumina

12. Site of flourophore cleavage
Blocking modification

13. STOPPED HERE

14. Limitations of Illumina sequencing
Slow run time
Short read lengths (~150 nt)
Fluorophore overlap
Out-of-phase accumulation