Genome sequencing

1. Genome sequencing MUPGRET Workshop Joe Polacco

2. Size of human genome • 23 pairs of chromosomes • 3.1 billion bp • If code written in NYC phone books and stacked up would reach top of Washington monument.

3. Human Genome Project • Began as a academic effort • Initially involved 5 research centers in US and England. • Soon joined by Celera, spin off company.

4. Some surprises • Initial estimate 100,000 to 150,000 genes but found to be 35,000 to 50,000. (C. elegans ~19,000 genes) • Mass of genome that codes for protein originally estimated as 5% but found to be 1.5%.

5. Some completely sequenced genomes • Mycoplasma genetialium • 578,000 bp, 400 genes • Haemophilus influenza • 1,830,138 bp, 1738 genes • E. coli • 4,639,221 bp, 4377 genes • S. cervisiae • 12 x 106 bp, 5885 genes

6. More genomes • C. elegans • 95.5 x 106, 19,820 genes • D. melanogaster • 1.8 x 108, 13,601 genes • A. thaliana • 1.17 x 108, 25, 498 genes

7. More genomes • M. musculus • 3 x 109, ~30,000 genes • H. sapiens • 3.3 X 109, 30-50,000 genes • O. sativa • 4.3 x 108, 30-63,000 genes

8. The beginning • Human genome project initially discussed at a UC-Santa Cruz meeting in 1985.

9. What were the concerns? • What will it do to biology? • How will be pay for it? • Is this really science? • Why bother to sequence it all? • all vs. just the genes (skim sequencing)

10. Dept. of Energy • Initially funded project in 1987. • $5.3 million • Study radiation induced mutations, repair and effect on humans.

11. NIH • Joined in 1988. • James Watson leader • 3% of research budget devoted to examining the ethical, legal, and social implications of gene research (ELSI)

12. Other genomes • Parallel sequencing of E. coli, S. cerevisiae, C. elegans, D. melanogaster, and M. musculus • Why • Work out the technology and methods

13. Watson’s vision • Sequence it all not just genes. • Use genetic maps and markers to help assemble the pieces.

14. Academic players • Wash U • Baylor • Whitehead • Wellcome Trust • Joint Genome Institute—DOE Center

15. $1 to 10 cents a finished bp • automated processing of cloned DNA • automated DNA sequencing • computer system to support sequence data • algorithms to assess sequence fidelity, assemble sequences, and “find” genes.

16. Maps • Thomas Hunt Morgan (early 1900s)—low resolution phenotypic markers • 1970s restriction maps • 1980s RFLPs • 1989 Maynard Olson, Leroy Hood, Charles Cantor, and David Botstein sequence itself is a marker! (STS)

17. PCR • Polymerase Chain Reaction • http://www.dnai.org/b/index.html • Techniques • Amplifying • Making copies of DNA

18. The PCR revolution • 1985 • Kary Mullis-Cetus Corporation • No need to send clones back and forth • Allowed automated DNA sequencing • No need for large clone repositiory for all human genes • Unrestricted access to genes via public sequence databases.

19. Kary Mullis talks about PCR • http://www.dnai.org/b/index.html • Techniques • Amplifying • Interviews • Making DNA copies • Naming PCR

20. Sequencing-the old way • Maxim and Gilbert or Sanger methods • http://www.dnai.org/b/index.html • Techniques • Sorting and Amplifying • Early DNA sequencing • http://www.dnai.org/b/index.html • Techniques • Sorting and Amplifying • Interviews • Dideoxy method of sequencing

21. Automated Sequencing • Automation made possible by new dye chemistry developed by Leroy Hood and Lloyd Smith at Cal. Inst. Tech. in 1986. • http://www.dnai.org/b/index.html • Techniques • Sorting and Amplifying • Cycle Sequencing

22. Inside the automated sequencer • Collaboration with ABI produced first automated sequencer. • Laser detection of each bp. • http://www.dnai.org/b/index.html • Techniques • Sorting and Amplifying • Interviews • Making sequencing automated • Inside an automated sequencer

23. Sequencing • Detect all 4 nucleotides in one lane so quadrupled the output from a single sequencing gel. • Dupont dye terminators—allowed all four nucleotides to be attached to terminal nucleotide in the same sequencing reaction. • Capillary eliminated need to cast gels.

24. Sequencing the Genome an Overview • Show sequencing.exe file containing movie about sequencing the human genome.

25. Two approaches to sequence the genome • Hierarchical Shotgun clone libraries • Use map to pick pieces of genome in order, break them, sequence and reassemble. (Watson) • Whole genome shotgun • Break up genomic DNA randomly, sequence several genome equivalents, and reassemble. (Ventner)

26. Hierarchical Shotgun Clone Libraries • Top-down strategy • Ordered library of clones based on large scale maps. • Subclone larger inserts into sequencing vector. • Reassemble sequence. • Based on order.

27. ESTs • Expressed sequence tags • Reverse transcribe mRNA and sequence. • Venter used nonspecific primer to randomly amplify 150-400 bp fragments of genes.

28. Patent controversy • NIH announced it would seed a patent on Venter’s STS. • Very controversial since functionally unknown. • More appropriate to private company. • Watson said it was “sheer lunacy” and resigned due to conflict with Bernardine Healy NIH director.

29. More patent • Many biotech companies arose at the time to mine ESTs and applied for patents on the genes for diagnostics and pharmaceuticals. • NIH withdrew patent application. • ESTs must be novel to be patented. • ESTs must be useful to be patented.

30. The result • No patents granted thus far on genes without known function.

31. Whole genome shotgun • Break the genome into a bunch of pieces often by mechanical shearing. • Sequence pieces and reassemble. • Weber (Marshfield Medical Research Foundation) and Myers (U of AZ) proposed method to speed sequencing. • 1998 Venter leaves NIH to head Celera and promised to sequence human genome in 3 years for $300 million.

32. Accelerated the public project. • Whole genome method was tested by sequencing 120 Mbp of Drosophila genome.