Presentation on theme: "Next–generation DNA sequencing technologies – theory & practice"— Presentation transcript:
1 Next–generation DNA sequencing technologies – theory & practice
2 Outline Next-Generation sequencing (NGS) technologies – overview NGS targeted re-sequencing – fishing out the regions of interestNGS workflow: data collection and processing – the exome sequencing pipeline
4 DNA Sequencing – the next generation The automated Sanger method is considered as a ‘first- generation’ technology, and newer methods are referred to as next- generation sequencing (NGS).
5 Landmarks in DNA sequencing 1953 Discovery of DNA double helix structure1977A Maxam and W Gilbert "DNA seq by chemical degradation"F Sanger"DNA sequencing with chain-terminating inhibitors"1984 DNA sequence of the Epstein-Barr virus, 170 kb1987 Applied Biosystems - first automated sequencer1991 Sequencing of human genome in Venter's lab1996 P. Nyrén and M Ronaghi - pyrosequencing2001 A draft sequence of the human genome2003 human genome completedLife Sciences markets first NGS machine
11 Template preparation: STEP1 Produce a non-biased source of nucleic acid material from the genome
12 Template preparation: STEP1 Produce a non-biased source of nucleic acid material from the genome
13 Template preparationProduce a non-biased source of nucleic acid material from the genomeCurrent methods:randomly breaking genomic DNA into smaller sizesLigate adaptorsattach or immobilize the template to a solid surface or supportthe spatially separated template sites allows thousands to billions of sequencing reactions to be performed simultaneously
26 DNA Sequencing – the next generation The major advance offered by NGS is the ability to cheaply produce an enormous volume of dataThe arrival of NGS technologies in the marketplace has changed the way we think about scientific approaches in basic, applied and clinical research
27 PART II: NGS targeted resequencing fishing out the regions of interest
28 Random genome sequencing The beginningRandom genome sequencing???Sanger sequencingTargetedbp
29 DNA Sequencing – the next generation Library/template preparationLibrary enrichment for targetSequencing and imaging
30 Target enrichment strategies Random genome sequencingHybrid CapturePCR basedSanger sequencing
44 PART III: NGS workflowdata collection and processing – the exome sequencing pipeline
45 Whole Exome Sequencing The human genomeGenome = 3GbExome = 30MbexonsProtein coding genesconstitute only approximately 1% of the human genomeIt is estimated that 85% of the mutations with large effects on disease-related traits can be found in exons or splice sites
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