1. BME 130 – Genomes Lecture 4 Sequencing technology II Next generation sequencing

2. Figure 4.2 Genomes 3 (© Garland Science 2007)

3. Figure 4.3a Genomes 3 (© Garland Science 2007)

5. Roche / 454

6. 454 Process Overview 1) Prepare Adapter Ligated ssDNA Library 2) Clonal Amplification on 28 µ beads 4) Perform Sequencing by synthesis on the 454 Instrument of ~250,000 molecules 3) Load beads and enzymes in PicoTiter Plate™

7. Key sequence = TCAG for identifying wells and calibration TACGTACG Flow Order 454 base calling

8. Limitations of Roche/454

9. Solexa / Illumina

11. Limitations of Solexa/Illumina

12. ABI/SOLiD

13. Limitations of ABI/SOLiD

14. Applications of high-throughput sequencing

15. Genome assembly

16. CHiP-Seq Johnson et al., Science 8 June 2007: Vol. 316. no. 5830, pp. 1497 - 1502

17. RNASeq Cloonan et al., Genome Biology 2008, 9:234