1. Genome Sequencing and Assembly
Rui Alves

2. Outline of the Talk Genomes Methods for Genome Sequencing
(Y)BAC-to-(Y)BAC (HSG)
Shotgun Sequencing
Primer walking
Optical mapping
Direct Reads
Polonies
High density reactors

3. What are genomes?

4. 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

5. What are genomes?
Sequencing and annotating a genome reveals the proteins, tRNAs, rRNAs that each organisms possesses to adapt to their environment

6. Why sequence genomes? AACTGGTCCAT Genome Sequence A, B, C, …, Z
Dictionary
Write/ Read books
ACTG
Genes/
Proteins
Understand/
manipulate biology
AACTGGTCCAT
Genome Sequence

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

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

9. (Y)BAC-to-(Y)BAC Sequencing
Same
Restriction
Enzyme

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

11. How sequencing works: The Sanger Method
Denaturation
Repeat with 4 different ddNTPs
ddNTPc
dNTPc

12. Identifying the sequence
A C T G G C T A

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

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

15. Shotgun Sequencing
Restriction Enzymes

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

17. Comparison (Y)BAC WGS p(Recombination of (Y)BACs) >>
Contamination
p(Recombination of plasmids)
Less demanding during assembly
Better Orientation of Scaffold Assembly
More demanding during assembly
Worst Orientation of Scaffold Assembly
Better Coverage for Repeats
Worst Coverage for Repeats

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

19. 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

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

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

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

23. Optical Mapping

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

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

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

27. Direct reads Bases have different emission spectra
Fix straight DNA into slide
run slide through spectophotometer
Identify each base

28. Direct Reads A C G T A C T G

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

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

31. Polonies N N N Polony Pol N N N Pol N N N N Pol N N N N Pol N N N Pol

32. Sequencing Polonies A CC
dATP
dCTP

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

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

35. How are genomes assembled?
Fragment Chromosome into manageable pieces
Take fragments
Feed them into computer programs
Assembled them onto scaffold
Fill in the gaps