“Today we are initiating an unending study of human biology. Whatever else [happens]….

1. Genome Sequencing & the Human Genome ProjectSpeaker- Joy Scaria Biological Sciences Group

2. “Today we are initiating an unending study of human biology. Whatever else [happens]…. It will be an adventure, a priceless endeavor” -Norton Zinder Comparisons Manhattan Project Apollo programme

3. Genome Size Human genome- 3 Billion nucleotides(3.2Gb) E.coli – 4.7 million Nucleotides E. coli genome – 300 pages of a 1000 page book D.melanogaster- 10 books (8 Chromosomes) Human – 200 books( 200, 000 pages)

4. Origin of the project No one is certain as to who first suggested the idea First serious proposal was by Robert Sinsheimer Renato Dulbecco who was in attendance told this to J.D. Watson at CSHL DOE Scientists were made aware of Sinsheimer’s proposal Charles Delisi Head of DOE’s Health & Environmental Research convened a metting and offered Los Alamos And Livermore labs to start the project

5. Celera Vs Government project The cDNA dispute and patent claims Craig Ventor Establishes Celera Celera database is paid while the other is free

6. Whose genome is being sequenced? • the first reference genome is a composite genome from several different people • generated from 10-20 primary samples taken from numerous anonymous donors across racial and ethnic groups Male or Female ???

7. Whose Money ?? Major participant is US government Totally 20 Groups from UK, Japan, France Germany and China are involved Celera is a private company

8. Human Genome Project Project goals are to ■ identify all the approximate 30,000 genes in human DNA, ■ determine the sequences of the 3 billion chemical base pairs that make up human DNA, ■ store this information in databases, ■ improve tools for data analysis, ■ transfer related technologies to the private sector, and ■ address the ethical, legal, and social issues (ELSI) that may arise from the project. Recent Milestones: ■ June 2000 completion of a working draft of the entire human genome ■ February 2001 analyses of the working draft are published

9. Satellite projects

11. GenomeSequencing Published/complete genomes = 173 Prokaryotic ongoing projects = 432 Eukaryotic ongoing genomes = 368 Total = 974 (data as on 20-07-04) Many more on the way

14. Draft First Sequence later

16. Developing overlapping genomic clone library

17. Cloning in Bacteria

18. Robotic Assembly for HTG Sequencing

19. Determining the purity of DNA To determine the DNA concentration, the eluted samples are transferred into specially designed quartz glass microplates and the absorbancy at 260 nm and 320 nm

20. Sequencing The robot is gripping a 384 well microplate (used for PCR or sequencing) from a 384 well MJ PTC 225 Tetrad Block.

21. Automated liquid handling The Hydras are used for all liquid handling steps, e. g. setting up the PCR reaction, the purification, the sequencing, etc.

22. Station Fridge. The fridge is for plates with contents (e. g. sequencing mix, PCR mix, etc.) which have to be stored at +4 °C. The door is opened and closed pneumatically

23. Capillary Array Electrophoresis (CAE). DNA samples are introduced into the 96-capillary array; as the separated fragments pass through the capillaries, they are irradiated all at once with laser light. Fluorescence is measured by a charged coupled device that acts as a simultaneous multichannel detector. (Inset circle at upper left: Closeup view of individual capillary lanes with separated samples.

24. Technical Parlance Raw sequence: Individual unassembled sequence reads, produced by sequencing of clones containing DNA inserts. Paired-end sequence: Raw sequence obtained from both ends of a cloned insert in any vector, such as a plasmid or bacterial artificial chromosome. Finished sequence: Complete sequence of a clone or genome, with an accuracy of at least 99.99% and no gaps BAC clone: Bacterial artificial chromosome vector carrying a genomic DNA insert, typically 100–200 kb. Most of the large-insert clones sequenced in the project were BAC clones Draft clone :A large-insert clone for which roughly half-shotgun sequence has been produced.

25. Predraft clone: A large-insert clone for which some shotgun sequence is available, but which does not meet the standards for inclusion in the collection of draft clones. Contig The result of joining an overlapping collection of sequences or clones. Scaffold: The result of connecting contigs by linking information from paired-end reads from plasmids, paired-end reads from BACs, known messenger RNAs or other sources. The contigs in a scaffold are ordered and oriented with respect to one another STS :Sequence tagged site, corresponding to a short (typically less than 500 bp) unique genomic locus

26. Common Sources of STSs Expressed sequence tags (ESTs) are short sequences obtained by analysis of complementary DNA (cDNA) clones. Complementary DNA is prepared by converting mRNA into double-stranded DNA and is thought to represent the sequences of the genes being expressed. Simple sequence length polymorphisms (SSLPs) are arrays of repeat sequences that display length variations. SSLPs that are polymorphic and have already been mapped by linkage analysis are particularly valuable because they provide a connection between genetic and physical maps. Random genomic sequences

27. SNP :Single nucleotide polymorphism RFLP: Restriction fragment length polymorphism

28. What does the project reveal ? Gene Numbers- Celera estimates 26000, Public project puts it as 3100 Genome is laden with repeat sequences But Puffer fish Genome virtually lacks any Human repetitive sequences are old and enfeebled While Mouse repeats are dynamic More than 1.4 million SNPs has been found Origin of genome- From evolutionary past Only 94 of 1,278 protein families are vertebrate specific

29. Why did evolution favor Males No sexuality in lower kingdoms Hermaprodites are perfect animals After all Why do we need males? Instead of being parasites on females why do they just go away?

30. Total number of genes 252 SRY is mapped on Y.p11.3 It is just 896bp

31. Question of Mitochondrial Eve Medical research Artificial intelligence and many more…

32. Next Step in Genomics • Transcriptomics involves large‑scale analysis of messenger RNAs (molecules that are transcribed from active genes) to follow when, where, and under what conditions genes are expressed. • Proteomics—the study of protein expression and function—can bring researchers closer than gene expression studies to what’s actually happening in the cell. • Structural genomics initiatives are being launched worldwide to generate the 3‑D structures of one or more proteins from each protein family, thus offering clues to function and biological targets for drug design. • Knockout studies are one experimental method for understanding the function of DNA sequences and the proteins they encode. Researchers inactivate genes in living organisms and monitor any changes that could reveal the function of specific genes. • Comparative genomics—analyzing DNA sequence patterns of humans and well‑studied model organisms side‑by‑side—has become one of the most powerful strategies for identifying human genes and interpreting their function.

33. References http://www.nature.com/genomics/human/papers/409860a0_fs_1.html http://www.celera.com/therapeutics/home.cfm?ppage=genomics http://vector.cshl.org/ http://www.ornl.gov/hgmis/ Nature : Vol.409, No.6822., 15 Feb 2001