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Lecture 12

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Queen's University
BIOL 205

Milestones in Sequencing In 2003 the Human Genome Project “completed” an initial draft sequence of the human genome after ~13 years and a cumulative cost of ~$300 million. 1990 $10/base 2003 $0.10/base Costs dropped sharply over the course of project due to improvements in sequencing technology especially replacing gels with capillary electrophoresis Sanger sequencing used for the Human genome Denature How was the human genome sequenced? Human genome 3 billion base pairs To ensure accuracy 10 fold coverage 600 bp of sequence possible in each run Requires 50 million Independent successful runs Human Genome Project : Government based! A. Individual chromosomes or Total genome DNA B. Digest or shear –Clone into Cosmids – Library C. Isolate DNA from each cosmid clone – RE Fingerprint 4 5 3 2.5 5 3 2.5 2 1 3.5 6 Clone 1 Clone 2 D. Feed fingerprint data into computer –look for overlaps 4 5 3 2.5 5 3 2.5 2 1 3.5 6 Each Bac is fingerprinted –insert size -allows removal of bad clones Craig Venter Developed shotgun sequencing Founded “The institute of genomic research”(TIGR) Founded Celera Genomics •sequenced his dog! •predicts that future will have everyone sequenced! •Sanger institute:Announcement 10,000 human genomes in next 3 years. Whole genome shotgun sequencing Strategy…sequence first, map later Whole Genome Sequencing What has the sequence of the Human genome told us? Less than 3% encodes exons of mRNAs! Humans have surprising few genes ~ 19-22,000 protein ~27,000 protein encoding genes encoding genes Mustard plant Genes are unevenly distributed 26,588 Open reading frames (# is dropping 2006 – 19-22,000 predicted genes ORFs – protein encoding genes 6,538 matched known genes (from previous work) 11,226 had homology –human or other organisms -ORTHOLOGS 8, 619 based on computer prediction Average ORF size ~ 27 kbp 42% of ORFs have no known or predicted function! 38% GC 45% repetitive sequence Many genes – arose by duplication (retrovirus?) mRNA DNA copy Jump back into Reverse genome!?! Transcription Ts (Ts) Paralog Pseudogene Original 19,000 gene Pseudogenes In the Human genome No Protein Functional Protein COMPARATIVE GENOMICS HUMAN COMPLEXITY: Gene Duplication  Protein Domains -increased # and complexity 60% of human protein-coding genes have multiple splice variants On average 3 splice variants per gene Comparative genomics ‘Volcanoes in the genome’ - February 12, 2009 A burst of genetic changes occurred around 10 million years ago in one of the ancestors we share with chimpanzees,with consequences that are being felt today. A new analysis of the genomes of macaques, orang-utans, chimpanzees and humans shows that DNA segments in this ancestor began to make duplicate copies at a very high rate aroundten million years ago, even though other mutation processes such as single letter changes were slowing. “There’s a big burst of activity that happenswhere genomes are suddenlyrearrangedand changed,” says Evan Eichler, of the Howard Hughes Medical Institute. He adds, “Because of the architecture of the human genome, genetic material is constantly being added and deleted in certain regions. These are really like volcanoes in the genome, blowing out pieces of DNA.” In Nature the team reportsthat this duplicationspree occurred before humans and chimpanzees diverged,around 6 million years ago, but after the divergence from orang-utans 12-16 million years ago and the divergence from macaques prior to that. Comparative Genomics: Pathogenic versus non-pathogenic strains K12 (common lab strain) 2007 Outbreak O157:H7 -5416 genes - 1387 genes not found in K12 genome K12 -4405 genes – 528 not found in O157:H7 New genes in O157:H7 = virulence factors including toxins cell invasion proteins, adherance proteins 2006 Outbreak Potential new targets for intervention! 25 October 2011 do:10.1038/478444a Crreoin2NeFeaeur Plague genome: The Black Death decoded Thgenoa6y odbaumrevns efoonEuosdk c s .ter EeCaaay
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