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Lecture 8

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Western University
Biochemistry 2280A
Christopher Brandl

Sequencing Genomes ● Sequencing a genome involves: ○ Creating a genomic DNAlibrary ○ Many independent sequencing reactions ○ Aligning the independent sequences into a continuous sequence Creating a Genomic DNALibrary ● Agenomic library is the collection of cloned DNAfragments that represent all of the DNAin an organism’s genome ○ each cloned DNAis like a different book in the library ● First, you get lot’s of the DNAisolated from many cells, and then cleave it with a restriction nuclease; this gives you millions of genomic DNAfragments ● With those DNAfragments, you clone them into a plasmid via a ligation reaction ○ first form recombinant DNAwith those fragments and next introduce them into the plasmids to allow for replication of each fragment ● Then the bacteria will of course divide and this results in millions of clones ● These millions of clones are your library as each cell contains a different fragment of DNAand the whole collection represents the entire genome For example, let’s go through sequencing the genomic library of H.influenzae: ● Start with billions of cells of the bacteria, and extract the DNAfrom the cells ● Now we want to break up the DNAinto fragments; you can use restriction enzymes but also sonification (using sound to break the DNA) ● Ideally you wanted ~2000 bp fragments; to do this, they ran the DNAthrough an electrophoresis gel, and cut out all the fragments that were around the size of 2000bp ● After purifying the DNAfrom the gel, you prepare a clone library: resulted in ~20 000 clones with each cell representing a fragment of the DNA ● Now you want to sequence the ends of the genomic clones; to do this they isolated the plasmid DNAfrom each cell, annealed a primer and then ● 25 000 sequence runs were made from 20 000 clones and this resulted in 12 million bp of sequence ○ Some of the end sequences were obtained from one side, others from both sides ● Now, you want to put all the 25 000 sequences together in the correct order (this is the hard part) ● What you do is get a computer search for overlaps between the 25 000 sequence runs and then fits them together into what are called contigs ○ a contig is a continuous DNAsequence representing a portion of the genome ■ a contig is not a clone or physical entity, it is an aligned sequence determined by the computer ● After the computer has aligned the sequences, it generates 140 contigs ○ one genome should be 1 contig, thus 140 contigs means there are some gaps missing ● With 140 contigs you will have 140 gaps in a circular genome; we need to fill in the gaps to order the sequence correctly ● There are two types of gaps: ○ sequence gaps (relatively easy to fill in) ○ physical gaps (not so easy) ● Sequencing gaps can be closed by completing the sequence of clones in the library ● The computer scans for original clones that are found in two different contigs ● What you do is sequence the cloned DNAfragment t
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