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Mapping and Sequencing Genomes

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Mapping and Sequencing Genomes

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    1. Mapping and Sequencing Genomes

    2. Sanger Sequencing

    3. Sanger Sequencing

    4. Sanger Sequencing-Critical Innovations

    5. BAC-by-BAC Sequencing

    6. Whole Genome Shotgun Sequencing

    7. Combined Approach

    8. Calculating Sequence Coverage

    9. Lander-Waterman Model Poisson Estimate Number of reads Average length of a read

    10. Poisson Distribution Digression

    11. Poisson is a good estimate for… Number of misprints per page in a book Number of traffic lights per mile of roadway Number of trout per cubic meter of pond water

    12. Poisson is specified by a single parameter, ?

    13. Poisson Distribution

    14. Poisson Distribution

    15. Back to Sequencing

    16. Lander–Waterman Assumptions

    21. Lander–Waterman Assumptions

    22. In practice… Lander-Waterman is almost always an underestimate -cloning biases in shotgun libraries -repeats -GC/AT rich regions -other low complexity regions

    23. Mapping/Ordering BACs

    31. When is a genome finished? 1) Finishing is hard! 2) Quality values: Phred score = -10*log10P(error) Phred20=1error/100bp How much continuous phred20 sequence? 3) Gaps? 1 contig/chromosome (probably not)

    32. EST Projects EST=Expressed Sequence Tag Short, single pass reads from bits of mRNA In practice random reads from cDNA libraries polyA primed/random primed Sometimes libraries are tissue specific

    33. ESTs Ups: Represent the part of the genome (most) people care about Does not require a sequenced genome Find genes Find SNPs Find splice isoforms

    34. What is the future of sequencing? Resequencing One human done?4 billion to go Locating polymorphisms for complex diseases More species, more individuals Comparative genomics What resolution (ORFs, transcription factors, individual base pairs) determines how many genomes

    35. Really high-throughput sequencing High-throughput=Cheap $50 million/per mammalian genome (now) Reduce volumes=reduce reagent cost Eliminate/parallelize cloning and DNA preparation Multiplex!

    36. The $1000 Genome

    37. Key ingredients so far… Sequencing by synthesis Elimination/parallelization of clone production miniaturization

    38. Pyrosequencing I

    39. Pyrosequencing II

    40. Illumina Sequencing

    41. Illumina Sequencing

    42. Illumina Sequencing

    43. ABI/SOLiD Sequencing

    44. ABI/SOLiD Sequencing

    45. Single Molecule Sequencing

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