2. DNA Sequencing – “Plus and Minus” Plus –Incubate with T4 DNA Polymerase and single dNTP –T4 Polymerase degrades 3’ ends in absence of dNTP –Fractionated on PAGE w/ladder Minus –Incubate with DNA Polymerase I and 3 dNTPs –Denatured from template –PAGE electrophoresis Disadvantage –Neither method completely accurate –Both must be used together

3. DNA Sequencing – Chemical Cleavage Maxam and Gilbert

4. DNA Sequencing – Dideoxy Sequencing DNA sequencing with chain-terminating inhibitors F. Sanger, S. Nickelen, and A.R. Coulson (1977)

5. Sanger et al Bacteriophage  X174 Restriction enzyme frag. –A12d and A14 ddGTP, ddATP, ddTTP and araCTP –Arabinonucleoside dATP chase

6. Sanger et al R4 fragment as primer Digest with restriction enzyme ddNTPs used Problem –What if second restriction site close to first one –New bands arise

7. Sanger et al A8 fragment as primer Single ribonucleotide incorporated at end of primer ddTTP, araCTP, ddCTP, ddGTP, and ddATP

8. Shotgun Sequencing Genomic DNA sheared Cloned into vectors Sequence Fragment  Contigs

9. Determining the sequence of both ends of the shotgun library End-sequencing large-insert clones to find one that spans the region of interest Shotgun Sequencing (cont.)

10. Pilot project for Human Genome 2 kbp and 10 kbp mate pairs BACs were end-sequenced to generate set of mate pairs Problem with assembly –Confusion with repetitive DNA stretches Solution – Scaffolds of contigs

11. Myers et al High quality input data needed Accuracy of pair mate distances Reliablity of true pairing The Drosophila Data Sets WGS – 2 kbp, 10 kbp and BAC Joint Data Set –WGS –2 nd and 3 rd chromosome –340 BAC and P1 inserts –X chromosome –4 th chromosome

12. Myers et al Screen for rep. elements –Ribosomal DNA –Histones –Heterochromatin –Retrotransposons Overlap: true/repeat induced

13. Myers et al Unitigger –Unique or repetitive seq –Correct subassembly or fragments –Detect repeat boundaries so can remove repetitive overlap between the unitigs

14. Myers et al Scaffolder Repeat Resolutions –Rocks - two mate pairs –Stones - single mate pair –Pebbles – find best tiling across gap Consensus

15. Myers et al