中科院bio2000课件 分子生物学-zhu-part1.pdf

High-Throughput Biology Genomics Transcription Functional Systems Genomics Biology Translation Proteomics   Genomics   Functional Genomics   Proteomics   Genomics What can happen at the DNA level?   Chromosome stability   Chromatin   Telomere   Deletion/Insertion   Rearrangement   Mutation   Methylation Whole Genome Sequencing Big Science! History in Genome Sequencing Steps in decoding a genome (old fashion): 1) Library construction 2) Physical Map (contig) construction 3) DNA sequencing strategies 4) Sequence Assembly 5) Finishing 6) Annotation Milestones: Saccharomyces cerevisiae : 1996, 12 Mbp, cosmid 9 Homo sapiens: 1990-2003, 10 bp Oryza sativa ssp. indica/japonica The Walking Method 1.  Build a very redundant library of BACs with sequenced clone-ends (cheap to build) 2.  Sequence some “seed” clones 3.  “Walk” from seeds using clone- ends to pick library clones that extend left right Walking: An Example Walking off several seeds in parallel Efficient Inefficient •  Few sequential steps •  Additional redundant sequencing In general, can sequence a genome in ~5 walking steps, with 20% redundant sequencing Using Two Libraries Most inefficiency comes from closing a small ocean with a much larger clone Solution: Use a second library of small clones Whole Genome Shotgun Sequencing genome cut many times at random plasmids (2–10 Kb)