Genome sequencing and assembling.ppt

Genome sequencing and assembling Chitta Baral Basic ideas and limitations Current lab techniques can sequence small (say 700 base pairs) DNA pieces. Use restriction enzymes to cut DNA pieces Sort pieces of different sizes using gel electrophoresis and use the sorting to read them Mapping and Walking Sequence one piece, get 700 letters, make a primer that allowed you to read the next 700, and work sequentially down the clone Estimate for human genome sequencing using this method: 100 years Shotgun sequencing (introduced by Sanger et al. 1977) for sequencing genomes Obtain random sequence reads from a genome Assemble them into contigs on the basis of sequence overlaps Straightforward for simple genomes (with no or few repeat sequences) Merge reads containing overlapping sequence Shotgun sequencing is more challenging for complex (repeat-rich) genomes: two approaches Shotgun sequencing – 2 approaches Hierarchical shotgun approach Generating an overlapping set of intermediate-sized (e.g. bacterial artificial chromosomes with 200 KB inserts) clones, and keeping a map of that (it took 2 yrs for mapping e-coli) Subjecting each of these clones to shotgun sequencing, and using the map to get the whole sequence. Used in S. cerevisiae (yeast), C. elegans (nematode), A. thaliana (mustard weed) and by the International Human Genome Sequencing Consortium (started in 1990, draft made available in 2000) Whole-genome shotgun (WGS) approach Generating sequence reads directly from a whole-genome library Using computational techniques to reassemble in one step. Used for Drosophila melanogaster (fruit fly) and by Celera Genomics (formed 1998) for human genome. Sequencing small DNA pieces Use DNA cloning or PCR to make multiple copies. Put in 4 testtubes marked G, A, T and C In testtube G use restriction enzymes that cuts at G. Do the above step for the other testubes. Use gel electrophoresis separately for the content in each testtube. The data results in the table on the left. Readin