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

2. Administrivia Syllabus change (see website) Groups should decide on paper to present by Thursday

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

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

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

7. Roche / 454

8. 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™

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

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

11. Solexa / Illumina

13. Limitations of Illumina sequencing Slow run time Shorter read lengths (~100 nt) Fluorophore overlap

14. ABI/SOLiD

15. Limitations of ABI/SOLiD Complicated color-space encoding Requires a close genome for mapping Short read lengths (~50 nt)

16. PlatformRead length (nt)Reads per runTotal sequence (nt) Roche/4544001,000,000400,000,000 Illumina100140,000,00014,000,000,000 ABI/SOLiD501,000,000,00050,000,000,000

17. Ion torrent