AB & BCI               AB Solid,                                 CE Sequencing         Illumina GA,    Séquencage         ...
AB & BCI               AB Solid,                                 CE Sequencing         Illumina GA,    Séquencage         ...
Virus: 3500 à 8 x 105 bases                               Bactéries plus de1Mb                            (Escherichia col...
Avant: le séquencage enzymatique= SANGER Sequencing   ADN simple brin + ADN polymérase   addition d ’un didéoxy.en petit...
Avant: le séquencage enzymatique= SANGER Sequencing   ADN simple brin + ADN polymérase   addition d ’un didéoxy.en petit...
Avant: entre gels plats et capillaires
Avant: entre gels plats et capillaires
Avant: entre gels plats et capillaires
Sanger vs NextGen
Cout séquencage:3+1+(0.4+4.5+0.4)x2=14.6€/1séq. ds de 700b      Idée duCEQ 8 capillaires: 33.000b ds/24h (48x2x700b)      ...
Roche GS-FLXti                                                                  0.4 Gb/runNext Generation                 ...
The Polonator G.007 is the                                                                                             fir...
Roche Applied-Science  GS-20, GS-FLX, GS-FLXti           (454)
• GS-FLXti Data                  DNA Library Preparation and Titration   emPCR    Sequencing                  4.5 h       ...
• GS-FLXti Data                  DNA Library Preparation and Titration         emPCR                   Sequencing         ...
•Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter re...
•Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter re...
•Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter re...
•Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter re...
•Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter re...
•Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter re...
DNA Library Preparation and Titration      emPCR                    Sequencing• GS-FLXti Data     4.5 h                   ...
T•Raw data is                       Cprocessed                     Gfrom a series             Aof individual       Timages...
•Raw data isprocessedfrom a seriesof individualimages.                   Key sequence = TCAG for identifying wells and cal...
• GS-FLXti Data   • Quality filtered bases                    400-500 bp average read length                    > 0.4 Gb o...
SoftwareSoftwareMapping               MappingImage     Signal      Sequence                Reference sequence             ...
• GS-FLXti Data                  Sanger: Weeks                  454: 4 days                       Sanger Technology       ...
NextGen                      Sequencers                                                                Roche GS-FLX:      ...
NextGen                      Sequencers                                                                Roche GS-FLX:      ...
NextGen                                                              Roche GS-FLX:          Sequencers           add-onsno...
Roche FLXti:Next Generation        0.5 Gb/run                    1m reads @ 400b  Sequencers            €5990/run         ...
illuminaGenome Analyzer    (Solexa)
Illuminas Solexa Sequencing Technology                    Step 1: Sample Preparation                    The DNA sample of ...
Illuminas Solexa Sequencing Technology                    Step 1: Sample Preparation                    The DNA sample of ...
Illuminas Solexa Sequencing Technology                    Step 1: Sample Preparation                    The DNA sample of ...
Illuminas Solexa Sequencing Technology                    Step 1: Sample Preparation                    The DNA sample of ...
Illuminas Solexa Sequencing Technology                    Step 1: Sample Preparation                    The DNA sample of ...
Illuminas Solexa Sequencing Technology                    Step 1: Sample Preparation                    The DNA sample of ...
Illuminas Solexa Sequencing Technology                    Steps 7-12: Sequencing by Synthesis                    A flow ce...
Illuminas Solexa Sequencing Technology                    Steps 7-12: Sequencing by Synthesis                    A flow ce...
Illuminas Solexa Sequencing Technology                    Steps 7-12: Sequencing by Synthesis                    A flow ce...
Illuminas Solexa Sequencing Technology                    Steps 7-12: Sequencing by Synthesis                    A flow ce...
Illuminas Solexa Sequencing Technology                    Steps 7-12: Sequencing by Synthesis                    A flow ce...
Illuminas Solexa Sequencing Technology                    Steps 7-12: Sequencing by Synthesis                    A flow ce...
Pipeline software highlightsAutomated image calibration: maximizes the number of clusters used to generate sequence dataAc...
Sanger: WeeksIllumina:   <7   days Technology Comparison Sanger vs. Solexa technology for a 2-Gigabase genome
Sanger: WeeksIllumina:   <7   days Technology Comparison Sanger vs. Solexa technology for a 2-Gigabase genome
NextGen                                                                              Illumina GA2:                Sequence...
NextGen                                                   Illumina GA2:       Sequencers        add-onsnot included - Clus...
Illumina GA2:Next Generation    5-10 Gb/run (50b)                  $8250 (€6180)/run (5Gb)  Sequencers              $0,33/...
Applied BioSystems      SOLID system  (Agencourt BioScience)
SOLiD v2 instrument componentsThe SOLiD™ Instrument consists ofthe following components:• Reagent delivery system• Electro...
Figure 1. Library generation schematic.Sequencing on the SOLiD machine starts with library preparation. In the simplestfra...
Figure 2. Clonal bead library generation via emulsion PCR.Once the adapters are ligated to the library, emulsion PCR is co...
Figure 3. Depositing beads into flow cell via end modifications.Each bead is then attached to the surface of a flow cell v...
Each oligo has degeneratepositions at 3’ bases 1-3(N’s), and one of 16 specificdinucleotides at positions4-5. Positions 6 ...
Each oligo has degeneratepositions at 3’ bases 1-3(N’s), and one of 16 specificdinucleotides at positions4-5. Positions 6 ...
For example (see Fig.4), the dinucleotidesCA, AC, TG, and GT areall encoded by thegreen dye.Because each base isqueried tw...
For example (see Fig.4), the dinucleotidesCA, AC, TG, and GT areall encoded by thegreen dye.Because each base isqueried tw...
NextGen                 AB Solid 3.0:Sequencers Workflow             Workflow: 3-4 days (setup) + 4-10 days (run)
NextGen                 AB Solid 3.0:Sequencers Workflow             Workflow: 3-4 days (setup) + 4-10 days (run)
NextGen                 AB Solid 3.0:Sequencers Workflow             Workflow: 3-4 days (setup) + 4-10 days (run)
NextGen                 AB Solid 3.0:Sequencers Workflow             Workflow: 3-4 days (setup) + 4-10 days (run)
NextGen                                                          AB Solid 3.0:            Sequencers             add-onsCo...
AB Solid 3.0Next Generation     10-20 Gb/run                  100m reads @ 50b                  €5300/run 5+5Gb  Sequencer...
Roche GS-FLXti:     Roche GS-FLXti:   0.5 Gb/run           Setup time: 3-4 d1m reads @ 400b             0.4Gb/run         ...
General                      Roche GS-FLXti  Infrastructure                                Laboratory 1    Controlled     ...
NextGen Sequencing ServiceProvidersEuropeMany locations             Cogenics                                       http://...
Whole genome           Amplicon seq.       Transcriptome seq.sequencing             - Mutations / SNP   - cDNA- de novo se...
AB:                                                     Roche1, 4, 8 regionsslides16-128 samples/slidewith barcoding      ...
Roche (192)                                  AB (256)    Illumina (96)Increase Sample Throughput   via Multiplex Identifiers
ABIllumina                        Paired-end                        Sequencing           Roche
Capture allhuman exons on                 Increase Selectivity via   7 chips           CHIP selection
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  • Nextgentechnologies 124159213386-phpapp01

    1. 1. AB & BCI AB Solid, CE Sequencing Illumina GA, Séquencage Roche-454 FLX Nouvelle Génération AB 96 capillaires: Roche:NextGen Sequencers - NextGen 2,8 Mb/24h 0,4 GB/4jSequencing - NGS 400b/read Illumina: BCI 8 capillaires: 10 GB/6jWhole Genome Sequencer - Whole 45KB/24h AB/LT:Genome Sequencing - WGS 700b/read 20 GB/10j
    2. 2. AB & BCI AB Solid, CE Sequencing Illumina GA, Séquencage Roche-454 FLX Nouvelle Génération AB 96 capillaires: Roche:NextGen Sequencers - NextGen 2,8 Mb/24h 0,4 GB/4jSequencing - NGS 400b/read Illumina: BCI 8 capillaires: 10 GB/6jWhole Genome Sequencer - Whole 45KB/24h AB/LT:Genome Sequencing - WGS 700b/read 20 GB/10j
    3. 3. Virus: 3500 à 8 x 105 bases Bactéries plus de1Mb (Escherichia coli = 4,7 Mb)Basics 1 kilobase 1kb = 1 000 bases Eucaryotes de 10 à 3 x 105 Mb levure = 1,3 Mb drosophile = 165 Mb 1 mégabase 1Mb 1 000 000 bases 1 million Homo sapiens 3400 Mb 3Gb 20 000-25 000 genes Transcriptome = 2% Genome 1 gigabase 1 Gb 1000 Mb 1 milliard
    4. 4. Avant: le séquencage enzymatique= SANGER Sequencing ADN simple brin + ADN polymérase addition d ’un didéoxy.en petite quantité (ddNTP) 4 réactions pour les 4 bases en //, chacune avec 1 didéoxy. différent synthèse arrêtée à chaque incorporation d ’un didéoxy. statistiquement, autant de fragments avortés que de fois où la base est représentée
    5. 5. Avant: le séquencage enzymatique= SANGER Sequencing ADN simple brin + ADN polymérase addition d ’un didéoxy.en petite quantité (ddNTP) 4 réactions pour les 4 bases en //, chacune avec 1 didéoxy. différent synthèse arrêtée à chaque incorporation d ’un didéoxy. statistiquement, autant de fragments avortés que de fois où la base est représentée
    6. 6. Avant: entre gels plats et capillaires
    7. 7. Avant: entre gels plats et capillaires
    8. 8. Avant: entre gels plats et capillaires
    9. 9. Sanger vs NextGen
    10. 10. Cout séquencage:3+1+(0.4+4.5+0.4)x2=14.6€/1séq. ds de 700b Idée duCEQ 8 capillaires: 33.000b ds/24h (48x2x700b) cout duCout séquencage de 33.000b ds: 688€Cout séquencage de 1Mb ds: 20.848€ CEquencingBioinformatique, confirmation:5min/1000b 7hrs/33.000b
    11. 11. Roche GS-FLXti 0.4 Gb/runNext Generation 1m reads @ 400b Sequencers €5990/run €14.97/Mb €500k/inst. Illumina GA2NextGen Sequencers - NextGen Sequencing(NGS) 5-10 Gb/run 60m reads @ 50bWhole Genome Sequencer - Whole GenomeSequencing (WGS) $8250(€6180)/run (5Gb) $0,33(€0,25)/Mb $460k(€344k)/inst. AB Solid 3.0 10-20 Gb/run 100m reads @ 50b €5300/run 5+5GbThe competition:Helicos Biosciences, Pacific Biosiences, George Church Lab., €0,53/MbNanopores sequencing, ZS-Genetics, Sequencing by TEM... €462k/inst.
    12. 12. The Polonator G.007 is the first "open source" gene sequencing instrument to hit Other Players the lab market in which the instruments software (Web ware) and specifications are freely available to the public. At $150,000, the Polonator is the cheapest instrument on the marketGeorge Church Lab. + Danaher Motion: Polonator G.007 The HeliScope™ Single Molecule Sequencer is the firstHelicos BioSciences Corp.: HeliScope SMS genetic analyzer to harness the power of direct DNA measurement, enabled by HelicosZS-Genetics: Electron Microscopy Sequencing. By the first True Single Moleculehalf of 2009, the system is expected to read complete a haploid Sequencing (tSMS)™human genome in approximately 8 days, with 4X coverage, at a cost technology.in the tens of thousands of dollars.Pacific BioSciences published technology for SingleMolecule Realtime Sequencing SMRT. Instrument by 2010Moebius Biosystems: Nexus. Over 6 Gigabases in 24hrs.Nanopore sequencing: Oxorf Nanopore, Sequenom...etc Pacific BioSciences
    13. 13. Roche Applied-Science GS-20, GS-FLX, GS-FLXti (454)
    14. 14. • GS-FLXti Data DNA Library Preparation and Titration emPCR Sequencing 4.5 h and 10.5 h 8h 10 h  Genome fragmented by nebulization  No cloning; no colony picking  sstDNA library created with adapters  A/B fragments selected using avidin-biotin purification gDNA sstDNA library Process Steps 1. DNA library preparation
    15. 15. • GS-FLXti Data DNA Library Preparation and Titration emPCR Sequencing 4.5 h and 10.5 h 8h 10 h Anneal sstDNA to Emulsify beads and Clonal amplification Break microreactors, an excess of DNA PCR reagents in water- occurs inside enrich for DNA- capture beads in-oil microreactors microreactors positive beads sstDNA library Clonally-amplified sstDNA attached to bead Process Steps 2. emulsion PCR
    16. 16. •Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter reactionvessels.•Each well is onlyable to accept asingle DNA bead. Load genome into Load PicoTiterPlate Load sequencing•Reactions in the PicoTiterPlate device device on instrument reagentswells are Close and Press GO! – sequence genomemeasured of theCCD camera. Process Steps•Titanium plate:3.4m wells 3. Sequencing with the PicoTiterPlate device
    17. 17. •Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter reactionvessels.•Each well is onlyable to accept asingle DNA bead. Load genome into Load PicoTiterPlate Load sequencing•Reactions in the PicoTiterPlate device device on instrument reagentswells are Close and Press GO! – sequence genomemeasured of theCCD camera. Process Steps•Titanium plate:3.4m wells 3. Sequencing with the PicoTiterPlate device
    18. 18. •Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter reactionvessels.•Each well is onlyable to accept asingle DNA bead. Load genome into Load PicoTiterPlate Load sequencing•Reactions in the PicoTiterPlate device device on instrument reagentswells are Close and Press GO! – sequence genomemeasured of theCCD camera. Process Steps•Titanium plate:3.4m wells 3. Sequencing with the PicoTiterPlate device
    19. 19. •Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter reactionvessels.•Each well is onlyable to accept asingle DNA bead. Load genome into Load PicoTiterPlate Load sequencing•Reactions in the PicoTiterPlate device device on instrument reagentswells are Close and Press GO! – sequence genomemeasured of theCCD camera. Process Steps•Titanium plate:3.4m wells 3. Sequencing with the PicoTiterPlate device
    20. 20. •Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter reactionvessels.•Each well is onlyable to accept asingle DNA bead. Load genome into Load PicoTiterPlate Load sequencing•Reactions in the PicoTiterPlate device device on instrument reagentswells are Close and Press GO! – sequence genomemeasured of theCCD camera. Process Steps•Titanium plate:3.4m wells 3. Sequencing with the PicoTiterPlate device
    21. 21. •Multiple opticalfibers are fused toform an opticalarray.•Proprietaryetching methodproduces wellsthat serve aspicoliter reactionvessels.•Each well is onlyable to accept asingle DNA bead. Load genome into Load PicoTiterPlate Load sequencing•Reactions in the PicoTiterPlate device device on instrument reagentswells are Close and Press GO! – sequence genomemeasured of theCCD camera. Process Steps•Titanium plate:3.4m wells 3. Sequencing with the PicoTiterPlate device
    22. 22. DNA Library Preparation and Titration emPCR Sequencing• GS-FLXti Data 4.5 h and 10.5 h 8h 10 h  3.4 m wells  3.4 m reads obtained in parallel  A single clonally amplified sstDNA bead is deposited per well. Amplified sstDNA library beads Quality filtered bases DNA capture  4 bases (TACG) bead containing cycled 200 times millions of copies  Chemiluminescent of a single clonal signal generation fragment  Signal processing to determine base sequence and quality score Amplified sstDNA library beads Quality filtered bases Process Steps 3. Sequencing
    23. 23. T•Raw data is Cprocessed Gfrom a series Aof individual Timages.•Each well’sdata isextracted,quantified, Metric and image viewing software Signal output from a single well (flowgram)andnormalized.•Read data isconvertedintoflowgrams. Process Steps 4. Signal-processing
    24. 24. •Raw data isprocessedfrom a seriesof individualimages. Key sequence = TCAG for identifying wells and calibration•Each well’s Flow of individual bases (TCAG) is 42 times.data is TAextracted, CGquantified,andnormalized. TTCTGCGAA•Read data isconvertedintoflowgrams. Base flow Signal strength Process Steps 4. Signal-processing
    25. 25. • GS-FLXti Data • Quality filtered bases 400-500 bp average read length > 0.4 Gb or 1m reads with a 70 x 75 mm FLXti PicoTiterPlate device 10 hours run time • Phred-like quality score for use in available assemblers or viewers • Consensus base-called contig files - FASTA file of assembled reads mapping against known scaffold (resequencing) de novo assembly of individual bases in consensus contigs • Viewer-ready genome file - assembly file in .ace format • Assembly metric files • Run-time metrics files - summarize important information pertaining to sequencing quality for each run Process Steps 5. Data output
    26. 26. SoftwareSoftwareMapping MappingImage Signal Sequence Reference sequence FlowMapper Software Reference sequence Fragments (reads)
    27. 27. • GS-FLXti Data Sanger: Weeks 454: 4 days Sanger Technology 7 days Weeks Preparation* Total Sequencing Time - DNA Library Preparation - 180 runs (1 per 4 hours) - Cloning - 2-million-base (Mb) genome - Template Preparation - 6x coverage 454 Technology 2.5 days 1 day Preparation Total Sequencing Time - DNA Library Preparation - 1 run (10 hours) - Titration of Library Beads - 400-600 million-base (Mb) - emPCR Technology Comparison Sanger vs. 454 technology for a 2-million-base genome
    28. 28. NextGen Sequencers Roche GS-FLX: Workflow Workflow 3-4 days (setup) + 1 day (run) IT steps: 1. Generation of a single-stranded template DNA library (~8-16 hours) 2. Emulsion-based clonal amplification of the library (~8 hours) 3. Data generation via sequencing-by-synthesis (9 hours) 4. Image and Base calling analysis (~8 hours) GS-FLX Software 5. Data analysis using different bioinformatics tools ▪GS Reference Mapper•Long Single Reads / Standard Shotgun (required input = 3–5μg,5μg recommended) ▪GS De Novo Assembler ▪GS Amplicon Variant Analyzer~1,000,000 single reads with an average read length of 400 bases •Paired End Reads (required input = 5μg @25 ng/μl or above, in TE; >10kb)◦3K Long-Tag Paired End Reads. Sequence 100 bases from each end of a 3,000 base spanon a single sequence read (Figure). Co-assemble GS FLX Titanium shotgun reads with 3KLong-Tag Paired Ends reads from Standard series runs. Third Party Software•Sequence Capture (required input = 3–5μg)◦Roche NimbleGen Sequence Capture using a single microarray hybridization-basedenrichment process.•Amplicon Sequencing (1-5ng or 10-50ng)◦The DNA-sample preparation for Amplicon Sequencing with the GS FLX System consists of asimple PCR amplification reaction with special Fusion Primers. The Fusion Primer consists of a20-25 bp target-specific sequence (3 end) and a 19 bp fixed sequence (Primer A or Primer Bon the 5 end).
    29. 29. NextGen Sequencers Roche GS-FLX: Workflow Workflow 3-4 days (setup) + 1 day (run) IT steps: 1. Generation of a single-stranded template DNA library (~8-16 hours) 2. Emulsion-based clonal amplification of the library (~8 hours) 3. Data generation via sequencing-by-synthesis (9 hours) 4. Image and Base calling analysis (~8 hours) GS-FLX Software 5. Data analysis using different bioinformatics tools ▪GS Reference Mapper•Long Single Reads / Standard Shotgun (required input = 3–5μg,5μg recommended) ▪GS De Novo Assembler ▪GS Amplicon Variant Analyzer~1,000,000 single reads with an average read length of 400 bases •Paired End Reads (required input = 5μg @25 ng/μl or above, in TE; >10kb)◦3K Long-Tag Paired End Reads. Sequence 100 bases from each end of a 3,000 base spanon a single sequence read (Figure). Co-assemble GS FLX Titanium shotgun reads with 3KLong-Tag Paired Ends reads from Standard series runs. Third Party Software•Sequence Capture (required input = 3–5μg)◦Roche NimbleGen Sequence Capture using a single microarray hybridization-basedenrichment process.•Amplicon Sequencing (1-5ng or 10-50ng)◦The DNA-sample preparation for Amplicon Sequencing with the GS FLX System consists of asimple PCR amplification reaction with special Fusion Primers. The Fusion Primer consists of a20-25 bp target-specific sequence (3 end) and a 19 bp fixed sequence (Primer A or Primer Bon the 5 end).
    30. 30. NextGen Roche GS-FLX: Sequencers add-onsnot included - Nebulizers + nitrogen tank Nebulization is required to shear fragments for DNA >70-800bp - emPCR Breaking Kit This device is required for the preparation of consistently sized reactors for emulsion PCR. - Magnetic Concentrator IVGN +€5000 - MT plate centrifuge BCI +€15.000 - Multisizer™ 3 COULTER counter +€15.000 The most versatile and accurate particle sizing and counting analyzer available today. Using The Coulter Principle, also known as ESZ (Electrical Sensing Zone Method), the Multisizer 3 COULTER COUNTER provides number, volume, mass and surface area size distributions in one measurement, with an overall sizing range of 0.4 µm to 1,200 - Agilent BioAnalyzer +€20.000 - Titanium cluster station +€29.000
    31. 31. Roche FLXti:Next Generation 0.5 Gb/run 1m reads @ 400b Sequencers €5990/run €14.97/Mb €585k/inst. tot The Roche Roche FLXti: Setup time: 3-4 d 0.5 Gb/run System Run time: 10 hrs images: 27 GB Primary Analysis: 15 GB PA CPU time: 80-220 hrs (6-7 hrs with cluster st) Final file size: 4 GB notes: 400-500b frag. length sequencing future dev. up to 1000b x coverage with long frag. vs x+n coverage with short reads vs cost/ Mb 10 systems in France ≈200 publications Multiplexing capacity
    32. 32. illuminaGenome Analyzer (Solexa)
    33. 33. Illuminas Solexa Sequencing Technology Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR. 1.5 days. Steps 2-6: Cluster Generation by Bridge Amplification In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is referred to as Illuminas "cluster station", an automated flow cell processor. 8hrs.
    34. 34. Illuminas Solexa Sequencing Technology Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR. 1.5 days. Steps 2-6: Cluster Generation by Bridge Amplification In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is referred to as Illuminas "cluster station", an automated flow cell processor. 8hrs.
    35. 35. Illuminas Solexa Sequencing Technology Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR. 1.5 days. Steps 2-6: Cluster Generation by Bridge Amplification In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is referred to as Illuminas "cluster station", an automated flow cell processor. 8hrs.
    36. 36. Illuminas Solexa Sequencing Technology Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR. 1.5 days. Steps 2-6: Cluster Generation by Bridge Amplification In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is referred to as Illuminas "cluster station", an automated flow cell processor. 8hrs.
    37. 37. Illuminas Solexa Sequencing Technology Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR. 1.5 days. Steps 2-6: Cluster Generation by Bridge Amplification In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is referred to as Illuminas "cluster station", an automated flow cell processor. 8hrs.
    38. 38. Illuminas Solexa Sequencing Technology Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR. 1.5 days. Steps 2-6: Cluster Generation by Bridge Amplification In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is referred to as Illuminas "cluster station", an automated flow cell processor. 8hrs.
    39. 39. Illuminas Solexa Sequencing Technology Steps 7-12: Sequencing by Synthesis A flow cell containing millions of unique clusters is now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases. The use of physical location to identify unique reads is a critical concept for all next generation sequencing systems. The density of the reads and the ability to image them without interfering noise is vital to the throughput of a given instrument. Each platform has its own unique issues that determine this number, 454 is limited by the number of wells in their PicoTiterPlate, Illumina is limited by fragment length that can effectively "bridge", and all providers are limited by flow cell real estate. 2-6 days (18-36 cycles).
    40. 40. Illuminas Solexa Sequencing Technology Steps 7-12: Sequencing by Synthesis A flow cell containing millions of unique clusters is now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases. The use of physical location to identify unique reads is a critical concept for all next generation sequencing systems. The density of the reads and the ability to image them without interfering noise is vital to the throughput of a given instrument. Each platform has its own unique issues that determine this number, 454 is limited by the number of wells in their PicoTiterPlate, Illumina is limited by fragment length that can effectively "bridge", and all providers are limited by flow cell real estate. 2-6 days (18-36 cycles).
    41. 41. Illuminas Solexa Sequencing Technology Steps 7-12: Sequencing by Synthesis A flow cell containing millions of unique clusters is now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases. The use of physical location to identify unique reads is a critical concept for all next generation sequencing systems. The density of the reads and the ability to image them without interfering noise is vital to the throughput of a given instrument. Each platform has its own unique issues that determine this number, 454 is limited by the number of wells in their PicoTiterPlate, Illumina is limited by fragment length that can effectively "bridge", and all providers are limited by flow cell real estate. 2-6 days (18-36 cycles).
    42. 42. Illuminas Solexa Sequencing Technology Steps 7-12: Sequencing by Synthesis A flow cell containing millions of unique clusters is now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases. The use of physical location to identify unique reads is a critical concept for all next generation sequencing systems. The density of the reads and the ability to image them without interfering noise is vital to the throughput of a given instrument. Each platform has its own unique issues that determine this number, 454 is limited by the number of wells in their PicoTiterPlate, Illumina is limited by fragment length that can effectively "bridge", and all providers are limited by flow cell real estate. 2-6 days (18-36 cycles).
    43. 43. Illuminas Solexa Sequencing Technology Steps 7-12: Sequencing by Synthesis A flow cell containing millions of unique clusters is now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases. The use of physical location to identify unique reads is a critical concept for all next generation sequencing systems. The density of the reads and the ability to image them without interfering noise is vital to the throughput of a given instrument. Each platform has its own unique issues that determine this number, 454 is limited by the number of wells in their PicoTiterPlate, Illumina is limited by fragment length that can effectively "bridge", and all providers are limited by flow cell real estate. 2-6 days (18-36 cycles).
    44. 44. Illuminas Solexa Sequencing Technology Steps 7-12: Sequencing by Synthesis A flow cell containing millions of unique clusters is now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases. The use of physical location to identify unique reads is a critical concept for all next generation sequencing systems. The density of the reads and the ability to image them without interfering noise is vital to the throughput of a given instrument. Each platform has its own unique issues that determine this number, 454 is limited by the number of wells in their PicoTiterPlate, Illumina is limited by fragment length that can effectively "bridge", and all providers are limited by flow cell real estate. 2-6 days (18-36 cycles).
    45. 45. Pipeline software highlightsAutomated image calibration: maximizes the number of clusters used to generate sequence dataAccurate cluster intensity scoring algorithms: allow efficient filtering for high-quality readsQuality-calibrated base calls: minimize the propagation of downstream sequencing errorsHighly optimized genomic alignment tools: minimize the need for elaborate computer infrastructuresOpen source code: enables researchers to customize the software to meet their needs
    46. 46. Sanger: WeeksIllumina: <7 days Technology Comparison Sanger vs. Solexa technology for a 2-Gigabase genome
    47. 47. Sanger: WeeksIllumina: <7 days Technology Comparison Sanger vs. Solexa technology for a 2-Gigabase genome
    48. 48. NextGen Illumina GA2: Sequencers Workflow Tracking ▪ Samples ready for sample prepWorkflow 2-3 days (setup) + 2-3 days (run) ▪ Samples ready for cluster prep 1. Non amplified DNA/RNA Sample ▪ Flow cells ready for sequencing 2. QC and possibly purify 3. Process with appropriate Sample Prep Kit 4. QC sample prep DAS2 server ▪Serve analysis files to DAS2 enabled genome 5. Assemble 7 samples with the same number of cycles, library browsers for direct visualization of results types, and sample types without file download 6. Process grouped samples with appropriate Cluster Generation Kit ▪Private server up and going using Authentication 7. Run cluster generation Mapping application (to handle 5-100 million 15-50bp sequences) 8. Transfer flow cell onto Genome Analyzer 9. Run sequencing 1st cycle ▪ Filter sequences by quality score 10. QC 1st cycle ▪ Count and remove identical sequences 11. Run remaining cycles ▪ Map sequences to reference genome 12. Export data 13. Run analysis Filter application ▪ Take binary map files and filter based on type of aligment and # of counts ▪ Export filtered universal binary for downstream applications Distributed Annotation System (DAS) defines a communication protocol used to exchange biological annotations
    49. 49. NextGen Illumina GA2: Sequencers add-onsnot included - Cluster Station +$50.000 The Cluster Station is a standalone, software- controlled system for the automated generation of clonal clusters from single molecule fragments on Illumina Genome Analyzer flow cells. - Paired-End Module +$45.000 The Paired-End Module provides fully automated template preparation for the second round of sequencing in a paired-end sequencing run. - IPAR +$60.000 IPAR is a bundled hardware and software solution that provides real-time quality control and integrated online processing of primary data during sequencing runs - Agilent BioAnalyzer +€20.000Total: €126.000
    50. 50. Illumina GA2:Next Generation 5-10 Gb/run (50b) $8250 (€6180)/run (5Gb) Sequencers $0,33/Mb €480/inst. tot The Illumina Illumina GA2: Setup time: 2-3 d 6-11 Gb/run System Run time: 3-6 d images: 900 GB Primary Analysis: 350 GB PA CPU time: 100 hrs Final File Size: 75 GB notes: 7/15 Gb by end of 2009 72 frag. length 9 systems in France 325 publications Multiplexing capacity
    51. 51. Applied BioSystems SOLID system (Agencourt BioScience)
    52. 52. SOLiD v2 instrument componentsThe SOLiD™ Instrument consists ofthe following components:• Reagent delivery system• Electronics• Camera (4 megapixel)• Monitor stand• Independently controlled dual flowcells• Liquid waste containerSOLiD v2 computer systeminstrument controller• Hardware: Intel® Xeon® processors• Operating system: Microsoft®Windows® XP Pro• Installed RAM: 4 GB• Hard disk storage: dual 80 GBSATA hard drives (RAID-1)head node• Hardware: Intel® Xeon® Dual Coreprocessors (2)• Operating system: 64-bit LINUX• Installed RAM: 8 GB• Hard disk storage: dual 750 GBSATA hard drives (RAID-1)compute nodes (each)• Hardware: Intel® Xeon® Dual Coreprocessors (2)• Operating System: 64-bit LINUX• Installed RAM: 8 GB SOLID in details• Hard disk storage: 80 GB SATA harddrivesstorage• Hard disk storage:15x 750 GB SATA hard drives• Operating system: 64-bit LINUX• RAID-5 w/ hot spare
    53. 53. Figure 1. Library generation schematic.Sequencing on the SOLiD machine starts with library preparation. In the simplestfragment library, two different adapters are ligated to sheared genomic DNA (leftpanel of Fig. 1). If more rigorous structural analysis is desired, a “mate-pair”library can be generated in a similar fashion, by incorporating a circularization/cleavage step prior to adapter ligation (right panel of Fig.1).ABIs SOLID SequencingTechnology
    54. 54. Figure 2. Clonal bead library generation via emulsion PCR.Once the adapters are ligated to the library, emulsion PCR is conducted using thecommon primers to generate “bead clones” which each contain a single nucleicacid species.ABIs SOLID SequencingTechnology
    55. 55. Figure 3. Depositing beads into flow cell via end modifications.Each bead is then attached to the surface of a flow cell via 3’ modifications to theDNA strands.At this point, we have a flow cell (basically a microscope slide that can be seriallyexposed to any liquids desired) whose surface is coated with thousands of beadseach containing a single genomic DNA species, with unique adapters on eitherend.Each microbead can be considered a separate sequencing reaction which ismonitored simultaneously via sequential digital imaging. Up to this point all next-gen sequencing technologies are very similar, this is where ABI/SOLiD divergesdramatically (see next).ABIs SOLID SequencingTechnology
    56. 56. Each oligo has degeneratepositions at 3’ bases 1-3(N’s), and one of 16 specificdinucleotides at positions4-5. Positions 6 through the5’ are also degenerate, andhold one of four fluorescentdyes. The sequencinginvolves: 1. Hybridization and ligation of a specific oligo whose 4th & 5th bases match that of the template 2. Detection of the specific fluor 3. Cleavage of all bases to the 5’ of base 5 4. Repeat, this time querying the 9th & 10th Figure 4. Schematic of ABI SOLiD sequencing chemistry. bases 5. After 5-7 cycles of this, perform a “reset”, in which the initial primer and all ligated portions are melted from the The actual base detection is no longer done by the polymerase-driven incorporation of template and labeled dideoxy terminators. Instead, SOLiD uses a mixture of labeled oligonucleotides discarded. and queries the input strand with ligase. Understanding the labeled oligo mixture is 6. Next a new initial key to understanding SOLiD technology. primer is used that is N-1 in length. Repeating the initial cycling (steps 1-4) now generates an overlapping data set ABIs SOLID Sequencing (bases 3/4, 8/9, etc, see Fig 5). Technology
    57. 57. Each oligo has degeneratepositions at 3’ bases 1-3(N’s), and one of 16 specificdinucleotides at positions4-5. Positions 6 through the5’ are also degenerate, andhold one of four fluorescentdyes. The sequencinginvolves: 1. Hybridization and ligation of a specific oligo whose 4th & 5th bases match that of the template 2. Detection of the specific fluor 3. Cleavage of all bases to the 5’ of base 5 4. Repeat, this time querying the 9th & 10th Figure 4. Schematic of ABI SOLiD sequencing chemistry. bases 5. After 5-7 cycles of this, perform a “reset”, in which the initial primer and all ligated portions are melted from the The actual base detection is no longer done by the polymerase-driven incorporation of template and labeled dideoxy terminators. Instead, SOLiD uses a mixture of labeled oligonucleotides discarded. and queries the input strand with ligase. Understanding the labeled oligo mixture is 6. Next a new initial key to understanding SOLiD technology. primer is used that is N-1 in length. Repeating the initial cycling (steps 1-4) now generates an overlapping data set ABIs SOLID Sequencing (bases 3/4, 8/9, etc, see Fig 5). Technology
    58. 58. For example (see Fig.4), the dinucleotidesCA, AC, TG, and GT areall encoded by thegreen dye.Because each base isqueried twice it ispossible, using the twocolors, to determinewhich bases were atwhich positions.This two color querysystem (known as Figure 5. Sequencing coverage during SOLiD sequencing cycles“color space” in ABI- Thus, 5-7 ligation reactions followed by a 4-5 primer reset cycles are repeatedspeak) has some generating sequence data for ~35 contiguous bases, in which each base hasinteresting been queried by two different oligonucleotides.consequences withregard to the If you’re doing the math you’ve realized there are 16 possible dinucleotidesidentification of errors. (4^2) and only 4 dyes. So data from a single color does not tell you what base is at a given position. This is where the brilliance (and potential confusion) comes about with regard to SOLiD. There are 4 oligos for every dye, meaning there are four dinucleotides that are encoded by each dye. ABIs SOLID Sequencing Technology
    59. 59. For example (see Fig.4), the dinucleotidesCA, AC, TG, and GT areall encoded by thegreen dye.Because each base isqueried twice it ispossible, using the twocolors, to determinewhich bases were atwhich positions.This two color querysystem (known as Figure 5. Sequencing coverage during SOLiD sequencing cycles“color space” in ABI- Thus, 5-7 ligation reactions followed by a 4-5 primer reset cycles are repeatedspeak) has some generating sequence data for ~35 contiguous bases, in which each base hasinteresting been queried by two different oligonucleotides.consequences withregard to the If you’re doing the math you’ve realized there are 16 possible dinucleotidesidentification of errors. (4^2) and only 4 dyes. So data from a single color does not tell you what base is at a given position. This is where the brilliance (and potential confusion) comes about with regard to SOLiD. There are 4 oligos for every dye, meaning there are four dinucleotides that are encoded by each dye. ABIs SOLID Sequencing Technology
    60. 60. NextGen AB Solid 3.0:Sequencers Workflow Workflow: 3-4 days (setup) + 4-10 days (run)
    61. 61. NextGen AB Solid 3.0:Sequencers Workflow Workflow: 3-4 days (setup) + 4-10 days (run)
    62. 62. NextGen AB Solid 3.0:Sequencers Workflow Workflow: 3-4 days (setup) + 4-10 days (run)
    63. 63. NextGen AB Solid 3.0:Sequencers Workflow Workflow: 3-4 days (setup) + 4-10 days (run)
    64. 64. NextGen AB Solid 3.0: Sequencers add-onsCovaris S2 System ULTRA-TURRAX TubeThe Covaris™ S2 System is required Drive from IKAsample preparation instrument for use This device is required for thein the SOLiD™ System workflow. The preparation of consistently sizedinstrument is an essential part of the reactors for emulsion PCR.emulsion PCR process used to preparethe beads for emulsion PCR. TheCovaris System is also used to shearDNA into 60 bp fragments for fragment Hydroshear fromlibrary preparation. Genomic Solutions The Hydroshear® from Genomic Solutions® is a reproducible and included controllable method for generating random DNA fragments of specific sizes. Use this to prepare mate- paired libraries for the SOLiD™ System.not included - Agilent BioAnalyzer +€20.000
    65. 65. AB Solid 3.0Next Generation 10-20 Gb/run 100m reads @ 50b €5300/run 5+5Gb Sequencers €0,53/Mb €482k/inst. tot The SOLID AB Solid 3.0: Setup time: 3-5 d 5-12.5 Gb/run/slide System Run time: 3.5-10 d images: 2.5 TB Primary Analysis: 750 GB PA CPU time: in run time Final file size: 140 GB notes: The Scientist Top Innovation of 2008 125-400m reads in 2009 30/40Gb potential for 12x human genome @ $10.000 3 systems in France Multiplexing capacity
    66. 66. Roche GS-FLXti: Roche GS-FLXti: 0.5 Gb/run Setup time: 3-4 d1m reads @ 400b 0.4Gb/run Run time: 10 hrs images: 27 GB €5990/run Primary Analysis: 15 GB €14.97/Mb PA CPU time: 220 hrs Final file size: 4 GB €585k/inst. tot Illumina GA2: Illumina GA2:5-10 Gb/run (50b) Setup time: 2-3 d 6-11 Gb/run€6180/run (5Gb) Run time: 3-6 d images: 900 GB €0,25/Mb Primary Analysis: 350 GB PA CPU time: 100 hrs €480/inst. tot Final File Size: 75 GB AB Solid 3.0 AB Solid 3.0: 10-20 Gb/run Setup time: 3-5 d100m reads @ 50b 5-12.5 Gb/run/slide Run time: 3.5-10 d€5300/run 5+5Gb images: 2.5 TB Primary Analysis: 750 GB €0,53/Mb PA CPU time: in run time Final file size: 140 GB €482k/inst. tot
    67. 67. General Roche GS-FLXti Infrastructure Laboratory 1 Controlled Room (emPCR) Amplicon Room Requirements BioIT room General Laboratory 2 Illumina GA2- Lab space, dedicated rooms General Laboratory 1- Hands on IT infrastructure Cluster Station room- Data Storage capacity General Laboratory 2 BioIT room-Sample and wor kflowtracability solutions General Laboratory 1 Controlled Room (emPCR) Amplicon- BioIT group support for 3rd Room General party analysis BioIT room Laboratory 2 AB Solid 3.0
    68. 68. NextGen Sequencing ServiceProvidersEuropeMany locations Cogenics http://www.cogenics.com/sequencing/s...ingService.cfmMany locations GATC Biotech http://www.gatc-biotech.com/en/index.phpGermany dkfz http://www.dkfz.de/gpcf/ngs_sequencing.htmlGermany Functional Genomics Center zurich http://www.fgcz.ethz.ch/applications/gt/ngsequencingGermany Eurofins MWG Operon http://www.eurofinsdna.com/products-...equencing.htmlHungary BAYGEN http://baygen.hu/The Netherlands ServiceXS http://www.servicexs.com/servicexs+i...+ii+sequencingSpain Sistemas Genómicos http://www.sistemasgenomicos.com/Sweden Sweden Uppsala Genome Center http://www.genpat.uu.se/node453Switzerland Fasteris http://www.fasteris.com/UK AGOWA - LGC http://www.lgc.co.uk/pdf/Next%20gen%...lyer%20web.pUK The Gene Pool https://www.wiki.ed.ac.uk/display/GenePool/HomeUK Geneservice http://www.geneservice.co.uk/services/sequencing/UK University of Liverpool http://www.liv.ac.uk/agf/index.htmlBelgium DNAVision (soon available) http://www.dnavision.be/ GATC Illumina platform based: 3500 € HT 1/8 flow cell vs 772 € Roche platform based: 10.150 € HT 1/2 picoplate vs 2995 € Cogenics 10/2008 Roche platform based: 15.000 € HT 1 full picoplate vs 5990 €
    69. 69. Whole genome Amplicon seq. Transcriptome seq.sequencing - Mutations / SNP - cDNA- de novo sequencing - Small RNA- comparative seq.Methylation seq. Metagenomics ChIP sequencing Les Applications
    70. 70. AB: Roche1, 4, 8 regionsslides16-128 samples/slidewith barcoding ABIllumina:2, 4, 8, regions Flow Cellflow cells – 1.4mm wide channel design – 40% more usable areaRoche:2, 4, 8, 16 regions Illuminaplates Multiple Sample Sequencing
    71. 71. Roche (192) AB (256) Illumina (96)Increase Sample Throughput via Multiplex Identifiers
    72. 72. ABIllumina Paired-end Sequencing Roche
    73. 73. Capture allhuman exons on Increase Selectivity via 7 chips CHIP selection
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