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Genome Sequencing and Assembly

Genome Sequencing and Assembly

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Genome Sequencing and Assembly

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  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

  7. 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

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

  14. 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

  15. Shotgun Sequencing Restriction Enzymes

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

  17. Comparison

  18. 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

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

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

  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 • 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. A C T G Direct Reads A C G T

  29. 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

  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 Pol N N Pol N N N N N N N N N N N Pol Pol N N N Pol

  32. Sequencing Polonies CC A dATP dCTP

  33. 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

  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