Next Generation Sequencing (NGS) Fernando Alvarez Sección Biomatemática, Facultad de Ciencias, UdelaR 1
Uruguay Montevide o
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TANGO World Champ 1930 1950 (Maraca 4
Next Generation Sequencing module Next Generation Sequencing technologies. Monday morning Genome Sequencing and Assembling. Monday afternoom Assembling Practical Monday afternoom RNA seq Theoretical and Practical. Tuesday morning Chiptser (Data analysis platform for microarray, proteomics and NGS data) Tuesday afternoom 5
Agenda 1- Sanger Sequencing 2- Next Generation Sequencing Deep sequencing Sanger sequencing Technologies Genome sequencers- Ultra high throughput sequencers - 454 Roche - Solexa illumina Description of the different technologies, comparison of their throughput, limitations 3- Next-next generation sequencers (single molecule sequencers) -Pacific Biosciences -Helicos 6
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DNA Sequencing 1977 Sanger enzymatic method dd(nucleotides) 8
DNA Sequencing Requirements: Template (to be sequenced) Primer dntp s ddntp s 4 independent reactions 9
DNA Sequencing Result: Fragments of different size according to where the ddntp has been incorporated Gel denaturing Gel separate the fragments to 10
DNA Sequencing Sequenced segment: 4040-3920 = bases 120 11
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Parallelization in the lab 13
Fluorometry and Sequencing 1986: contribution of fluorometry. Use of ddntp s linked to fluorescent labels Requeriments: Template Primer dntp s ddntp s (each one linked to a different Dye) Only 1 reaction Automatic Sequencing Nature 321, 674-679 (12 June 1986) 14
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Last 90 Applied Biosystems ABI 3730XL 96 capilaries 800 nt per read 1 Mb / days 16
Capillary sequencers (Sanger Centre, 2000) 17
Agenda 1- Sanger Sequencing 2- Next Generation Sequencing Deep sequencing Sanger sequencing Technologies Genome sequencers- Ultra high throughput sequencers - 454 Roche - Solexa illumina Description of the different technologies, comparison of their throughput, limitations 3- Next-next generation sequencers (single molecule sequencers) -Pacific Biosciences -Helicos 18
ROCHE 454 454 Sequencing is a massively-parallel pyrosequencing system capable of sequencing roughly 20 megabases of raw DNA per 7-hour (GS20). The GSFLX is able to sequence 100 Mb. The system relies on fixing nebulized and adapter-ligated DNA fragments to small DNA-capture beads in a water-in-oil emulsion. The DNA fixed to these beads is then amplified by PCR. Finally, each DNA-bound bead is placed into a ~44 μm well on a PicoTiterPlate, a fiber optic chip. A mix of enzymes such as polymerase, ATP sulfurylase, and luciferase are also packed 19
454 sequencer ROCHE 20
Amplification empcr (Emulsion PCR) 1 DNA fragment 1 bead 1 microreactor 21 One Fragment = One Bead (limiting concentration)
1,6 millones de secuenciadores 22
Pyrosequencing 23 23
Colección de Datos http://www.youtube.com/watch?v=nffgwgfe0aa&feature=related 24
Pyrogram 1st cycle 2nd cycle AGGGG TGGC 25
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Some Data 27
Some Data 2011: Genome Sequencer FLX System GS Junior System 28
Some throughput Data Genome Sequencer FLX System 29
Solexa Technology (200 Gb sequencer) 30
Deep sequencing 31
250 millions of clusters, 1000 copies each 32
33 http://seq.molbiol.ru/ Cesped de primers
http://seq.molbiol.ru/ 34
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Illumina 2011: 36
Illumina HiSeq1000-2000 37
Illumina miseq 38
Comparison of the technologies Technology Read Length Number of Reads Total throughput Capillary (Sanger) 800 nt 96 <80 kb 400 1.6 millions Roche (from 300 to 500) Solexa Solid 76 (100) 35 600 Mb Aprox 100 millions 10 Gb (x2) (x2) (today 200 GB ) 500 millions 16 GB 39
Agenda 1- Sanger Sequencing 2- Next Generation Sequencing Deep sequencing Sanger sequencing Technologies Genome sequencers- Ultra high throughput sequencers - 454 Roche - Solexa illumina Description of the different technologies, comparison of their throughput, limitations 3- Next-next generation sequencers (single molecule sequencers) -Pacific Biosciences -Helicos 40
Next-Next Generation Sequencers Sequencing without amplification Oxford Nanopore 41
PACBIO HELICOS 42
Secuenciación 2010-2011: J. Rothberg 43
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Ion Torrent 2011 PGM: 45
Ion Torrent 2011 PGM: 46
Secuenciación 2011 PGM: 47
Oxford Nanopore Tech: Oxford Nanopore Tech: 48
Oxford Nanopore α-hemolysin Staphylococcus aureus exonuclease 49
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Huma sequencing project (finished) 13 years, 3 dólares 2008 3 months, 60 k dolar, Today, 1 week 10 k dolars In the neext few years Few hours or minutes, less than 1000 dolars -Genome sequencing will become a routine clinical technique This is allowing and will allow even more, to pose questions that were unthinkable few years ago -Eg. what is the genetic basis of longevity, hypertension, or whatever trait with a very complex genetic basis 51
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Many things can be done, and many more will be possible in the future, 55