Genome Sequence determination

1. Genome Sequence determination 陳中庸 E-mail: cychen@cycu.edu.tw Web site: www.cychen.idv.tw

2. Complete Microbial Genomes

3. Genome what now? • Sequencing is… • Determining the full nucleotide sequence of one strain of an organism • Making predictions of genes within that sequence & predicting the function of those genes • HARD!!!! • Sequencing requires… • Time • Money • People • Computers

4. Genome what now? • Before Sequencing … • Nature of an organism • Genetic code • Genome size • Genome structure • Sequencing means… - Bioinformatic - Functional Assay - More….

5. Organism Selection Library Creation

6. Organism Selection Library Creation Sequencing

7. Organism Selection Library Creation Sequencing Assembly

8. Organism Selection Library Creation Sequencing Assembly

9. Organism Selection Library Creation Sequencing Assembly

10. Organism Selection Library Creation Sequencing Assembly Gap Closure

11. Organism Selection Library Creation Sequencing Assembly Gap Closure

12. Organism Selection Library Creation Sequencing Assembly Gap Closure Finishing

13. Organism Selection Library Creation Sequencing Assembly Gap Closure Finishing Annotation

14. Organism Selection Library Creation Sequencing Assembly Gap Closure Finishing Which steps are computationally expensive? Annotation

15. Organism Selection Library Creation Sequencing Assembly Gap Closure Finishing Annotation

16. Organism Selection Library Creation Sequencing Assembly Gap Closure Finishing Which steps have not already been exceptionally well studied? Annotation

17. Organism Selection Library Creation Sequencing Assembly Gap Closure Finishing Annotation

18. Organism Selection Library Creation Sequencing Assembly Gap Closure Finishing Which step has not been subjected to a variety of approaches? Annotation

19. Organism Selection Library Creation Sequencing Assembly Gap Closure Finishing Annotation

20. Organism Selection • Nature of an organism: Pathogen? • Genetic code • Genome size • Genome structure

21. Vibrio vulnificus Strain: YJ016 Genome Size: 5.2 Mb Source: Southern Taiwan Significance: Virulence Strategy: Whole Genome Shotgun Sequencing Coverage: 10X

22. Organism Selection • Nature of an organism: Pathogen? • Genetic code: Special Code? • Genome size • Genome structure

23. Genetic Code Tables http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c

24. Organism Selection • Nature of an organism: Pathogen? • Genetic code: Special Code? • Genome size: How many Megabases? • Genome structure

25. Organism Selection • Nature of an organism: Pathogen? • Genetic code: Special Code? • Genome size: How many Megabases? • Genome structure: Linear/Circular Chromosome? How many?

26. How to sequence a complete genome? Sizes of bacterial genomes vary between :Mycoplasma genitalium and Myxobacteria: 0.6 Mb to ~13 Mb • reading length of DNA sequencing reactions is just ~600 bp (= 0.0006 Mb) ⇒ a subdivision of the genome is obviously necessary • If the genome needs to be subdivided into small pieces of suitable sizes for sequencing, then • Individual sequences/fragments need to be ordered somehow into their "native" order • Therefore, overlaps between each other are necessary in order to re-assemble the pieces ⇒ there are two main sequencing strategies: 1. whole genome shotgun sequencing 2. ordered shotgun sequencing

27. c = Coverage;

28. Two ends are overlapped • Non overlapped • Plasmid percentage in contigs

30. Library Creation • Team Works • QC control • Time Table • Budget • Paper

31. Standard Operation Procedures of a Genome project A. Decision Mapping Protocol 1 QC PCR Confirm Protocol 2 B. Library Protocol 3 DNA purification Protocol 4 PFG QC FISH 決定盤數 PCR confirm Protocol 5 Shotgun Library Picking Print Labels C. Sequencing QC Protocol 6 Plasmid DNA Sequencing Reactions Dye Primers Protocol 7 QC Dye Terminator Protocol 8 Gel Running Protocol 9 377 QC Protocol 10 3700 D. Finish Protocol 11 Assemble Protocol 12 Annotation

32. Library (1) Random Shearing of Genomic DNA • Restriction enzyme: • Sau3AI (GATC)--- affected by CG methylase • MboI (GATC) – affected by dam methylase • -- not affected by CG methylase • 2. Sonication: • Sonication – Bal31 repair – T4 DNApolymerase – Sizing – • Recover –Ligation • 3. GeneMachine: easy sizing by filter

34. Library (2) Library clones & Sequencing clones Chromosome I Chromosome II 3.3 Mb 1.8 Mb Shotgun library Library 1: 2.5-3.5 kb inserts 7X Coverage Library 2: 5.5-7.5 kb inserts 3X Coverage Library 3: 30 kb inserts Cosmid library 10X Clone Coverage, 0.4X Sequence Coverage Sequenced for both ends Sequenced for both ends Sequenced for both ends Assemble the reads by using phred/phrap/consed softwares Contig 1 Contig 2 Contig 3 Closing the gaps by primer walking, PCR or re-sequencing Annotation

35. Library (2) Library clones & Sequencing clones 5,000,000 bp 1000 bp/per clone 5,000,000/1000 = 5000 clones =52 x 96 well plates 10 x redundancy 52 x10 x 96 wells plates Library clones Both ends sequencing 2 x 52 x 10 x 96 well plates ≒ 1000 plates Sequencing clones

36. Sequencing (1) Time table • 377:2 runs/per day (one run for one 96 well plate) • 3700 : 6 runs/per day (POP6) • 8 runs/per day (POP5) • 3730 : 12 runs/per day 2. 377 x 2 sets = 4 runs/per day 3700 x 2 sets = 6 x1 + 8 x 1 = 14 runs/per day total 18 runs per day 3. 1000 plates / 18 = 56 days = 11 weeks (3 months) 4. Today, 3730 for 4 sets = 48 runs/per day; 1000 plats /48 = 20 days

37. Sequencing (2) Cost

38. 硬體設施 ABI 377 ABI 3700 MegaBace 4000 ABI 3730XL

39. The automated production line for sample preparation at the Whitehead Institute, Center for Genome Research. The system consists of custom-designed factory-style conveyor belt robots that perform all functions from purifying DNA from bacterial cultures through setting up and purifying sequencing reactions.

40. 5X coverage 359 328 279 Assembled contigs 245 243 166 Assembled reads Reads vs. Assembled Contigs

41. 5X coverage Assembled size (Mbps) 5.17 5.12 5.13 5.10 5.08 5.07 Assembled reads Reads and Assembled Size

42. How assemble software works?

47. What is Gap Closure? • What are gaps? • Unsequenced regions located between assembly generated fragments of contiguous sequence (contigs) • What causes gaps? • Host toxicity, secondary structure, ??? • Back to “gap closure” • Producing, purifying, and sequencing, or locating, the missing regions of DNA

48. How Can I Close Gaps? • Genome Walking • Blind PCR extension of contigs • Multiplex PCR • Combinatorial trial of every contig pair • Read Pair Analysis • Use information stored by the assembler to suggest alignments, then PCR • Comparative Alignment

49. Comparative Alignment(the Bioinformatics Approach) • Find locations where contigs are homologous to known sequences • Determine if any contigs share homology in the same region of the same sequence • Design primers • Conduct PCR with those primers • Sequence that product and use that sequence to close the gap

50. Blast Organism X(cross) - Comparison • Compares contig ends to NCBI “nr” database with BlastN • Parses all hits and finds biologically possible contig pairs • Using the flanking sequence and Primer3, designs primers that will produce a PCR product spanning that gap