Le séquençage à très haut débit: développements et applica4ons dans les domaines de la Recherche et la Santé Laurent FARINELLI 17 November 2016 Journées Suisse Romande de forma;on Laboratoire et médecine SILAMED 2016 Lausanne www.fasteris.com
20 10 Fasteris years years of DNA illumina clusters NGS Celebra(ng 20 years of DNA clusters: Probably the longest experience with illumina NGS Celebra(ng 10 years of illumina NGS: Fasteris was the first service provider in the world to acquire an illumina instrument www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 2
DNA and PCR? • Who is familiar with DNA and PCR? ? ! www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 3
Nucleus versus Ball There is 2 meters of DNA in a cell Nucleus: 0.003 mm There would be 200 km in a basket ball Basket ball: 300 mm www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 4
Human genome length 150 000 000 km If human DNA was uncoiled and set end-to-end, it would represent 140 (mes the distance Earth - Sun www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 5
DNA vs RNA Base 5’ 3’ 5’ O P 3’ 5’ 3’ OH DeoxyriboNucleic Acid Base O P 3’ 5’ 3’ 5’ OH OH RiboNucleic Acid www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 6
DNA Replica4on DNA polymerase: 1 Unidirectional 5’ g 3’ 2 Requires a template 3 Requires a primer Tools: - PCR - DNA Sequencing www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 7
Liga4on: « colle » moléculaire DNA ligase: P P 3’ 5’ 5’ 3’ 1 Colle des brins P 2 A besoin de 5’-phosphate 3’ 5’ 5’ 3’ Outils: - Clonage d’ADN www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 8
Ciseaux moléculaires Enzyme de Restriction : 5’ -TACCGAATTCTAGG- 3’ 3’ -ATGGCTTAAGATCC- 5’ 1 Séquence reconnue Outils: - Clonage d’ADN 5’ -TACCG AATTCTAGG- 3’ 3’ 5’ 3’ -ATGGCTTAA GATCC- 5’ 3’ 5’ Nucleases non-spécifiques : 3’ 5’ 5’ Endonucleases 3’ Exonucleases www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 9
Ou4ls de Biologie Moléculaire Clonage d’ADN Polymerase Chain Reac(on (PCR) Séquençage d’ADN www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 10
Clonage d’ADN www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 11
Polymerase Chain Reac4on (PCR) Amplifica4on de l’ADN Polymérase (enzyme) www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 12
Polymerase Chain Reac4on (PCR) Cycle 1 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ Cycle 3 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ Cycle 2 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 5’ 3’ 3’ 5’ … n cycles => 2 n copies www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 13
Electrophorèse sur gel d’agarose www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 14
DNA sequencing? • Who is familiar with Sanger sequencing? ? ! www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 15
Séquençage d’ADN Méthode Sanger 5’ O Base 5’ P O O O 3’ OH O O Di DeoxyriboNucleic Acid 3’ é www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 16
Sanger Sequencing GCGAATGCGTCCACAAC G GCGAATGC G ddGTP GCGAAT G GCGAATGCGTCCACAACGCTACA GC G G GCGAATGCGTCCACAACGC T ddTTP G T A C GCGAATGCG T GCGAA T GCGAATGCGTCCACAACGCTAC A GCGAATGCGTCCACAACGCT A GCGAATGCGTCCACA A ddATP GCGAATGCGTCCAC A GCGAATGCGTCC A GCGA A GCG A GCGAATGCGTCCACAACGCTA C GCGAATGCGTCCACAACG C GCGAATGCGTCCACAA C ddCTP GCGAATGCGTCCA C GCGAATGCGTC C GCGAATGCGT C GCGAATG C G C www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 17
Sanger Sequencing GCGAATGCGTCCACAAC G GCGAATGC G ddGTP GCGAAT G GCGAATGCGTCCACAACGCTACA GC G G GCGAATGCGTCCACAACGC T ddTTP G T A C GCGAATGCG T GCGAA T GCGAATGCGTCCACAACGCTAC A GCGAATGCGTCCACAACGCT A GCGAATGCGTCCACA A ddATP GCGAATGCGTCCAC A GCGAATGCGTCC A GCGA A GCG A GCGAATGCGTCCACAACGCTA C GCGAATGCGTCCACAACG C GCGAATGCGTCCACAA C ddCTP GCGAATGCGTCCA C GCGAATGCGTC C GCGAATGCGT C GCGAATG C G C www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 18
Sanger Sequencing GCGAATGCGTCCACAAC G GCGAATGC G ddGTP GCGAAT G GCGAATGCGTCCACAACGCTACA GC G G G T A GCGAATGCGTCCACAACGC T ddTTP C GCGAATGCG T GCGAA T GCGAATGCGTCCACAACGCTAC A GCGAATGCGTCCACAACGCT A GCGAATGCGTCCACA A ddATP GCGAATGCGTCCAC A GCGAATGCGTCC A GCGA A GCG A GCGAATGCGTCCACAACGCTA C GCGAATGCGTCCACAACG C GCGAATGCGTCCACAA C ddCTP GCGAATGCGTCCA C GCGAATGCGTC C GCGAATGCGT C GCGAATG C G C www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 19
Automated Sanger Sequencer www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 20
NGS DNA sequencing? • Who is familiar with NGS? ? ! www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 21
Massively parallel sequencing Instead of reading one page at a (me We start reading one leaer at a (me, from each page simultaneously! www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 22
Massively parallel sequencing Sanger sequencing • One run in 1-2 hours • Read lengths of 800-1100 bases • Up to 96 (384) sequences in parallel Next-genera(on sequencing • One run in 1-2 hours to more than 6 days ! • Read lengths of 50-300 bases or even up to 40 kb • From 1 million to 5 billions sequences in parallel • A lot of diversity depending on the instruments www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 23
Massively parallel sequencing Single molecules or clonal amplifica(on? Either: • Amplify signal first • Amplify signal at later stage, during image acquisi(on A basic rule of physics: Amplify signal as soon as possible, else you will amplify more and more noise www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 24
Single Molecules Simpler sample prep Longer reads But also • Higher challenge • Lower accuracy • Challenge of detec(on (requires highly sensi(ve detectors, which are ofen fragile and/or costly) • Expect lower throughput www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 25
454 Corpora4on Based on pyrosequencing, developed in Sweden by Pål Nyhren and Mostafa Ronaghi GS - FLX GS - Junior www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 26
454 Corpora4on GS - FLX GS - Junior www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 27
Thermo Fisher • Ion S5 2.5 h run for 100 bases, 4h for 400 bases Three chips • Ion 520 , 1 - 2 Gb Ion S5 • 3-5 million reads • 200 or 400 bases reads • Ion 530 , 3 - 4 Gb • 15-20 million reads • 200 or 400 bases reads • Ion 540 , 10 - 15 Gb • 60-80 million reads • 200 bases reads www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 28
Pacific BioSciences www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 29
Oxford Nanopore Based on single-molecule sequencing Designed hand-held device MinION • Priced for USD 1000.- • Can sequence 2000 nanopores at a (me • Launched in 2015 www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 30
Everything started in 1996 in Geneva.. With the invention of DNA Colonies.. ..at GlaxoWellcome “Geneva Biomedical Research Ins(tute” www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 31
The DNA Colonies .. 1996-1997: GlaxoWellcome ’s Geneva Biomedical Research Institute Mayer P., Farinelli L. and Kawashima, E., 1997, Patent applica(on WO 98/44151 Key technology for illumina sequencing ...now know as DNA Clusters www.fasteris.com 17 November 2016 Laurent Farinelli – SILAMED 2016, Lausanne (c) 2016, Fasteris SA 32
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