DNA sequencing the beginnings Ghent University (Fiers et al) pioneers sequencing first complete gene (1972) first complete genome (1976) Next Generation Sequencing Fred Sanger develops dideoxy sequencing in 1975 Paul Coucke Center Medical Genetics Ghent Fred Sanger and Walter Gilbert receive Nobel prize in 1980 DNA sequencing the beginnings DNA sequencing the beginnings DNA sequencing the beginnings Automated DNA sequencing 1
The human genome project initiated in 1990 (James D. Watson) competition between public project and private company (Celera) HGP: 3,000,000,000 $ Celera: 300,000,000 $ 10 years of sequencing publication of a draft human genome Nature. 2001 Feb 15;409(6822):860-921 Science. 2001 Feb 16;291(5507):1304-51 Craig Venter, Bill Clinton and Francis Collins June 26 th, 2000 HGP Celera Where to go from here? Next generation sequencing A new sequencing technology is born method of the year next generation sequencing selected as method of the year 2007 by Nature Methods 2
Comparing technologies Roche sample prep Sanger: 1 reaction per sequence NGS: 1 reaction per run reading the sequence Sanger: size separation after sequencing reaction NGS: read during sequencing reaction throughput per run Sanger: <100 sequences NGS: millions of sequences emulsion PCR pyrosequencing picotiterplate 1M 450 bp reads 500 Mb / 10 hour cost Sanger: >1 / sequence NGS: <0.025 / sequence Pyrosequencing 3
454 LifeSciences Sequencer 1,6 million slots 454 sequencing Roche 454 sequencing Roche 120 miljoen pictures in 4 hours 4
flowgram 454 LifeSciences Sequencer A Jigsaw puzzle of the genome Bio-informatics is the bottleneck!!! Jigsaw puzzle with 1 million pieces... and some of them won t fit correctly Al 5
Illumina Solexa sequencing Illumina cluster PCR with bridge amplification single base extension sequencing 150M 100bp reads 12 Gb / 8 days Prepare DNA fragments Ligate adapters Attach single molecules to surface Amplify to form clusters Sequence Overview of sequencing technologies Applications for NGS company (instrument) from method read length throughput / run time Sanger 1977 chain termination sequencing 750 bp 50 Kb / 1 uur Roche (GS-FLX) 2005 pyrosequencing 400 bp 500 Mb / 10 uur Illumina (GA-II) 2006 sequencing by synthesis 100 bp 12 Gb / 8 dagen Applied Biosystems (SOLiD) 2007 ligation based sequencing 50 bp 20 Gb / 12 dagen Dover (Polonator) 2008 polymerase colony sequencing by synthesis 26 bp 10 Gb / 3 dagen Complete Genomics combinatorial probe-anchor ligation on 2008 (service) DNA nanoarrays 35 bp 6 Gb / dag Helicos (Heliscope) 2008 single molecule sequencing by synthesis 30 bp 25 Gb / 8 dagen Pacific Biosciences? 2010 single molecule real time sequencing by synthesis? 4000 bp? 100 Gb / 1 uur players and performance subject to quick changes many new approaches under development Genetic diagnostics using NGS technology Genetic diseases with multiple genes involved (genetic heterogenous disorders) - Congenital Hearing loss (> 20 genes) - BRCA1/2 (2 genes) - RP (> 15 genes) - Usher ( 9 genes) - Aortic Aneurysms (8 genes) -... Genetic diagnostics using NGS technology Major challenge is capturing the DNA of interest: -PCR amplification to capture sequences of interest (for example all exons of a set of genes) 6
Genetic diagnostics using NGS technology Genetic diagnostics using NGS technology BRCA1/2 genes (112 amplicons) - 16 multiplex PCR reactions - each amplicon has a patient specific ID-label (DNA sequence of 10 nucleotides incorporated during PCR) - Roche 454 : 100 patients / single run (one day) - sequencing cost : 200 Euro / patient Major challenge is capturing the DNA of interest: -PCR amplification to capture sequences of interest (for example all exons of a set of genes) - Microarray-based capture NimbleGen sequence capture Arrays - specific chromosomal region - whole exome - group of genes -... new opportunities for NGS in the clinic Personal genomics Personalised medicine & pharmacogenetics Challenges -management and analysis of massive amounts of genetic information -identification and interpretation of the thousands of variants within each individual genome -evaluation of the clinical relevance of identified variants Questions to society Is our society ready for personal genomics and personal healthcare? What is the cost of personal healthcare? How should we educate people to allow them to deal with this valuable but sensitive information? How do we protect people from genetic discrimination? Do our ethical principles need refinement to deal with the imminent genomic revolution? -how to deal with variants giving only marginal phenotypic effects 7
http://nxtgnt.com/ Be aware that most of what you heard will change rapidly! Thank you. 8