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2. Bioinformatics Prof:Rui Alves ralves@cmb.udl.es 973702406 Dept Ciencies Mediques Basiques, 1st Floor, Room 1.08 Website of the Course:http://web.udl.es/usuaris/pg193845/Courses/Bioinformatics_2007/ Course: http://10.100.14.36/Student_Server/

3. What is Bioinformatics? • Development and application of computational/informatic tools to the solution of biological problems • The Standard of internet Bioinformatics: MY DEFINITION LAM P E R L I N U X P A C H E Y S Q L H P Y T H O N Operating system Programing language(s) Internet server Database server

4. The standards are changing • JAVA facilitates that the servers launch a smaller number of processes by using the client’s machines for calculus and allowing for a larger number of simultaneous connections. • TOMCAT “talks” very well with JAVA. LTM J A V A I N U X O M C A T Y S Q L Operating system Programing language(s) Internet server Database server

5. Typical problems addressed • Genome Assembly and Gene Discovery & Tools • Data bases: Organizing and Storing Information • Sequence Alignments and Comparison Tools • Molecular Evolution & Tools • Analyzis of High Throughput Genomic/ Proteomic/ Metabolomic Experiments & Tools • Structural Analysis/Prediction & Tools • Mathematical Modelling & Tools • Integrating different tools/datasets • Development of Publicly available tools

6. Goals of the Course • Learn how to use computational tools to address problems at different biological levels • Learn to look for available tools • Learn to integrate different tools to solve a problem • Learn about the methods behind the tools • Learn to sort of know what to use depending on the problem at hand

7. Covered Material : Biological Perspective • Genome Sequencing and Assembly • Genome annotation • Protein function • Integrative analysis of the genome • Molecular Evolution • Systemic Molecular Biology • Ecosystems

8. Covered Material : Computational Perspective • Genome Assembly and Gene Discovery Tools • Data bases: Organizing and Storing Information • Sequence Alignments and Comparison Tools • Molecular Evolution Tools • Tools to Analyze High Throughput Genomic Experiments • Structural Analysis/Prediction Tools • Mathematical Modelling Tools • Integrating different tools • Publicly available tools

9. Evaluation: • 50% of the grade is given by the tasks you will perform along the semester • 40% of the grade will come from your final paper • 10% will be decided by me based on your overall performance. • CAUTION: You need to have passing grade oin both the tasks and th final paper to pass the course.

10. Genome sequencing and assembly Prof:Rui Alves ralves@cmb.udl.es 973702406 Dept Ciencies Mediques Basiques, 1st Floor, Room 1.08

11. Who has genomes?

12. What are genomes? • A genome is the whole native DNA content within a cell for any given organism • Genomes code for all proteins an organism needs to survive

13. How are genomes sequenced and assembled? • Fragment Chromosome(s) into manageable pieces and sequence these fragments • Take sequence of fragments • Feed them into computer programs • Assembled them onto scaffold • Fill in the gaps

14. How sequencing works: The Sanger Method Denaturation Add Polymerase + Nucleotides Repeat with 4 different ddNTPs ddNTPc dNTPc

15. Identifying the sequence A C T G Seq Smaller fragments will migrate faster … G … C … T … A …

16. Alternative sequencing methods: Pyrosequencing DNA A C T G G T C A DNAPol

17. Methods for Genome Sequencing • Methods for Genome Sequencing • (Y)BAC-to-(Y)BAC (HSG) • Shotgun Sequencing • Primer walking • Optical mapping • Polonies • High density reactors

18. (Y)BAC-to-(Y)BAC Sequencing Restriction Enzymes Replication (Yeast) Bacterial Artificial Chromosome

19. (Y)BAC-to-(Y)BAC Sequencing Sequence the edges Same Restriction Enzyme

20. (Y)BAC-to-(Y)BAC Sequencing Sequence fed into computer program

21. ACT…GTC CTA …ATC … …GGGG Computer assembly

22. Methods for Genome Sequencing • Methods for Genome Sequencing • (Y)BAC-to-(Y)BAC (HSG) • Shotgun Sequencing • Primer walking • Optical mapping • Polonies • High density reactors

23. Shotgun Sequencing Restriction Enzymes

24. ACT…GTC CTA …ATC … …GGGG Computer assembly

25. Comparison

26. Methods for Genome Sequencing • Methods for Genome Sequencing • (Y)BAC-to-(Y)BAC (HSG) • Shotgun Sequencing • Primer walking • Optical mapping • Polonies • High density reactors

27. Primer Walking • Sequencing an initial fragment. • Use the end of this sequence to design a primer • Primer will capture next fragment to be sequenced • Repeat until the end of the chromosome is reached

28. ACT…GTC CTA …ATC … …GGGG Primer Walking

29. New challenges in genome sequencing • Many organisms have been sequenced Finding intraspecific variations (e.g. SNPs, CNVs) Study evolution of strains • Faster & Cheaper sequencing methods are needed • These should also be able to deal with single polymeric DNA molecules

30. Methods for Genome Sequencing • Methods for Genome Sequencing • (Y)BAC-to-(Y)BAC (HSG) • Shotgun Sequencing • Primer walking • Optical mapping • Polonies • High density reactors

31. Optical Mapping

32. R1 R2 Ri Optical Mapping Original Genome Sequence Strain 1 Strain n

33. Determining the new sequence • Combining the know original genome sequence with Restriction Enzymes specificity predicts mutation • Small scale re-sequencing of appropriate regions

34. Methods for Genome Sequencing • Methods for Genome Sequencing • (Y)BAC-to-(Y)BAC (HSG) • Shotgun Sequencing • Primer walking • Optical mapping • Polonies • High density reactors

35. Polonies • Polonies are colonies of PCR amplicons derived from a single molecule of nucleic acid

36. Polonies N N Polony N Pol N N N Pol N N N N Pol N N N N Pol N N Pol N N Pol N N N N N N N N N N N Pol Pol N N N Pol

37. Sequencing Polonies CC A dATP dCTP

38. Methods for Genome Sequencing • Methods for Genome Sequencing • (Y)BAC-to-(Y)BAC (HSG) • Shotgun Sequencing • Primer walking • Optical mapping • Direct Reads • Polonies • High density reactors

39. High density reactors Fragments bound to beads, 1 per bead PCR of single DNA molecules (106 clones) Beads with imobilized enzymes for pyrophosphate sequencing

40. How are genomes assembled? • Fragment Chromosome into manageable pieces and sequence these pieces • Take sequence of fragments • Feed them into computer programs • Assembled them onto scaffold • Fill in the gaps

41. DNA Sequencing and assembly in a Nutshell • http://en.wikipedia.org/wiki/DNA_sequencing • http://en.wikipedia.org/wiki/Genome_assembly#Genome_assembly