High throughput sequencing
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High throughput sequencing

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  • 1. High throughput DNA sequencing Cosentino Cristian, PhD Genomics and Bioinformatics unit Filarete Foundation – Milan cosentia@gmail.com
  • 2. Summary 1 Classical sequecning method (Sanger) 2 Next-generation sequencing methods Roche 454 ABi SOLiD Illummina GAII 3 High throughput data management 4 High throughput sample preparation 6 Next-NGS sequencing Helicos HeliScope
  • 3. Summary 1 Classical sequecning method (Sanger) 2 Next-generation sequencing methods Roche 454 ABi SOLiD Illummina GAII 3 High throughput data management 4 High throughput sample preparation 6 Next-NGS sequencing Helicos HeliScope
  • 4. Approaching to NGS 2010 2000 1980 1990 1977 Sanger sequencing method by F. Sanger (PNAS ,1977, 74: 560-564) 1983 PCR by K. Mullis (Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73) 1953 Discovery of DNA structure Human Genome Project (Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31) (Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351) 1993 Development of pyrosequencing (Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365) Single molecule emulsion PCR 1998 Founded Solexa 1998 Founded 454 Life Science 2000 454 GS20 sequencer 2005 (First NGS sequencer) Solexa Genome Analyzer 2006 (First short-read NGS sequencer) Illumina acquires Solexa 2006 (Illumina enters the NGS business) ABI SOLiD 2007 (Short-read sequencer based upon ligation) Roche acquires 454 Life Sciences 2007 (Roche enters the NGS business) GS FLX sequencer 2008 (NGS with 400-500 bp read lenght) NGS Human Genome sequencing 2008 (First Human Genome sequencing based upon NGS technology) Hi-Seq2000 2010 (200Gbp per Flow Cell)
  • 5. Sequencing technologies DNA sequencing Classical approach Next-generation sequencing Individual sequencing reaction Massive parallel sequencing Clonally amplified DNAs Single molecule DNA (NGS) (N-NGS) Helicos Sanger method Illumina GAII HeliScope ABI SOLiD Roche 454 Output Output Single sequence ranging from 500 to 1000 bp Gbp of sequences ranging from 25 to 500 bp High Throughput
  • 6. Sanger method Sanger method with labeled dNTPs - The Sanger mehtods is based on the idea that inhibitors can terminate elongation of DNA at specific points Roche 454 ABi SOLiD Illumina GAII HeliScope Nanopore
  • 7. Summary 1 Classical sequecning method (Sanger) 2 Next-generation sequencing methods Roche 454 ABi SOLiD Illummina GAII 3 High throughput data management 4 High throughput sample preparation 6 Next-NGS sequencing Helicos HeliScope
  • 8. Next-generation sequencing platforms Isolation and purification of target DNA Sample preparation Library validation Amplification Cluster generation Emulsion PCR on solid-phase Sequencing Sequencing by synthesis Sequencing by synthesis with 3’-blocked reversible Pyrosequencing Sequencing by ligation with 3’-unblocked reversible terminators terminators Imaging Four colour imaging Single colour imaging Data analysis Illumina GAII Roche 454 ABi SOLiD Helicos HeliScope
  • 9. Roche 454 Pyrosequencing Sanger method - ABi SOLiD Illumina GAII HeliScope Nanopore
  • 10. Roche 454 ABi SOLiD Sample preparation Sanger method Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Nature Reviews genetics, 2010, 11: 31-46 - Discarded - fragments Illumina GAII HeliScope Nanopore gDNA gDNA Fragments are end- Fragments are denatured ssDNA 1:1 with agarose beads fragmented by repaired and ligated to and AB ssDNA are selected carrying oligos complementary nebulization or adaptors containing by avidin/biotin purification to adaptor sequences: 1 DNA sonication universal priming sites (ssDNA library) molecule/bead
  • 11. Roche 454 ABi SOLiD Emulsion PCR Sanger method Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Nature Reviews genetics, 2010, 11: 31-46 - Emulsified bead and PCR reagents into water-in-oil microreactors - Illumina GAII HeliScope Nanopore Clonal amplification inside microreactors Emulsion is disrupted and beads containing amplified template are enriched (1 million copies of templates/bead) Beads covalently Beads arrayed into linked to glass surfaces PicoTiterPlates ABi SOLiD Roche 454
  • 12. Roche 454 Pyrosequencing Sanger method Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Nature Reviews genetics, 2010, 11: 31-46 - 44 µm ABi SOLiD Illumina GAII HeliScope Nanopore Pyrosequecning Reads are recorded as flowgrams
  • 13. ABi SOLiD Sequecning by ligation Sanger method Roche 454 - Illumina GAII HeliScope Nanopore
  • 14. ABi SOLiD Di-base probe encoding system Sanger method Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Roche 454 - Illumina GAII HeliScope Nanopore
  • 15. ABi SOLiD Sequecning by ligation Sanger method Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Roche 454 - Illumina GAII HeliScope Nanopore 5 Universal Prime rounds (n to n-4), each with 7 probe ligations: 35 bp reads
  • 16. ABi SOLiD Colour encoding Sanger method Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Roche 454 - Illumina GAII HeliScope Nanopore Base zero is known
  • 17. ABi SOLiD Base zero Sanger method Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Roche 454 - Illumina GAII HeliScope Nanopore
  • 18. Illumina GAII Sequecning by synthesis with reversible terminator Sanger method Roche 454 ABi SOLiD - HeliScope Nanopore
  • 19. Illumina GAII Instrumentation Introduction Sample preparation Bioanalyzer 2100 Cluster station Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Paired-end module Genome Analyzer IIx Linux server
  • 20. Illumina GAII GAII applications Introduction Sample preparation Clusters amplification Application Source Sequencing by synthesis De novo gDNA sequencing gDNA Analysis pipeline Whole-genome resequencing gDNA High throughput Target resequecning Target enriched DNA sequences mRNA-seq Total RNA small RNA-seq Total RNA CHiP-seq Chip-DNA fragments Sequencing modes: • Single-read • Paired-end • Multiplexing
  • 21. Illumina GAII Sequencing workflow Introduction Sample preparation and Sample library validation preparation Clusters amplification Wash cluster station Cluster generation Sequencing by Cluster station synthesis Clusters Analysis amplification pipeline Linearization, High Blocking and throughput primer Hybridization Read 1 SBS sequencing GAIIx & PE Prepare read 2 Read 2 Pipeline base call Analysis Data analysis
  • 22. Illumina GAII Library preparation Introduction Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput gDNA Fragmented Adaptor- Double strand DNA ligated DNA denaturation
  • 23. Illumina GAII Library validation Introduction gDNA Sample preparation library Clusters amplification Sequencing by synthesis Analysis pipeline High throughput smallRNA library Bioanalyzer 2100
  • 24. Illumina GAII Cluster generation Introduction Sample preparation Clusters Validated library amplification Sequencing by synthesis Cluster station Analysis washing pipeline Cluster generation High Load reagents on throughput Cluster Station Cluster station Load DNA on Cluster Station Weekly manintenance wash Amplification on Cluster Station Linearization, Blocking and primer Hybridization SBS sequencing onto GAIIx
  • 25. Illumina GAII Flow cell Introduction Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput
  • 26. Illumina GAII Bridge amplification Introduction Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Hybridize adapter-ligated forward fragment and extend Extension is completed Denature dsDNA and wash original forward template; reverse template stays covalently attached to the array
  • 27. Illumina GAII Bridge amplification Introduction Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Bridge amplification of the reverse fragment Double-strand bridge is formed Double strand bridge is denatured and reverse as wel as forward fragments are covalentrly attached to the array
  • 28. Illumina GAII Bridge amplification Introduction Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Bridge amplification is repeated to enlarge the cluster Double-strand bridges are denatured Reverse strands fragments are cleaved and washed away
  • 29. Illumina GAII Bridge amplification Introduction Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Cluster with forward strands only, covalently attached to the array Sequencing primers start the SBS process
  • 30. Illumina GAII Sequencing with GAIIx Introduction Single read Cluster amplified Sample FlowCell preparation Clusters Wash GA & PEM amplification Sequencing by synthesis Install prism Analysis pipeline Install flow-cell High throughput Apply oil GAIIx First-base incorporation Adjust focus Check quality Weekly metrics manintenance wash 36-100 cycles Real-time sequencing run monitoring for Read 1/2 Post-run wash Analysis pipeline Linux server
  • 31. Illumina GAII SBS technology Introduction Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput
  • 32. Illumina GAII GAIIx optical path Introduction Sample preparation Clusters amplification Sequencing by synthesis Two colour excitation Analysis Four colour emission detection pipeline High throughput
  • 33. Illumina GAII Paired-end sequencing workflow Introduction Paired-end Sample preparation Wash GA & PEM Clusters amplification Sequencing by Install prism synthesis Analysis Install flow-cell pipeline High throughput Apply oil First-base GAIIx incorporation Adjust focus PEM Prepare Read 2 Check quality metrics 36-100 cycles Real-time sequencing run monitoring for Read 1/2 Post-run wash Analysis pipeline Linux server
  • 34. Illumina GAII Paired-end technology Introduction Single-read (read 1) Sample preparation Mapped Unmapped Clusters read read amplification Reference Gene 1 Gene 2 sequence Sequencing by synthesis Analysis pipeline Sequence reads High throughput Paired-end (read 1 & read 2) Mapped Mapped read read Reference Gene 1 Gene 2 sequence Sequence reads
  • 35. Illumina GAII Paired-end technology Introduction Paired-end sequencing works into GA and uses chemicals from the PE Sample module to perform cluster amplification of the reverse strand preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput
  • 36. Illumina GAII Firecrest and CASAVA Introduction Image files Intensity files Base calls files Sample preparation Firecrest Bustard From image From intensity Clusters amplification to intensity to reads Sequencing by synthesis Analysis pipeline High throughput Gerald Reads alignment Assembly Alignment files CASAVA Consensus assembly
  • 37. NGS technologies comparison Sequencing Amplif. Chemistry Read Run Gbp/ DNA $/sequencer lenght time day required (ref. 2008) (bp) (d) (μg) Roche 454 GS FLX emPCR Pyrosequencing 250- 0.35 1.3 3-5 500.000 Titanium 400 * ABi SOLiD emPCR Sequencing by 25-50 7-14 3.6 0.1-20 595.000 ligation Illumina GAII Solid-phase Reversible 36-100 4-9 3.9 0.1-1 430.000 terminator * Average
  • 38. NGS technologies comparison Sequencing Advantages Disadvantages $/Mbp (in 2008)* Roche 454 •Long reads even > 400 bp, •High indel in homopolymer 60 improving de novo sequencing stretches > 6 nucl. •Rare sustitution errors •High reagent cost •Longest reads only in single- read (2x150 bp) ABi SOLiD •Error correction with the two-base •Long time run 2 encoding system •Needs of cluster station to perform base calling and up to 1 week to align •Alignment must be performed against a reference db Illumina GAII •Most widely used platform (> 90 •Low multiplexing capability 2 science/nature publication) •Substitution errors •Sample preparation automatable •SBS , real-time analysis and base calling are performed simultaneously to the run •Automated cluster generation *Nat. Biotech., 2008, 26: 1135-1145
  • 39. Summary 1 Classical sequecning method (Sanger) 2 Next-generation sequencing methods Roche 454 ABi SOLiD Illummina GAII 3 High throughput data management 4 High throughput sample preparation 6 Next-NGS sequencing Helicos HeliScope
  • 40. Illumina GAII High throughput data storage Introduction Genotyping units Sample preparation Clusters amplification 0.5 – 14 GB/beadChip Sequencing by synthesis Analysis pipeline High throughput Data storage Tape recording unit Data storage Sequencing unit for offline backup mangement 200 Tb storage capacity 1 – 6 Tb/FlowCell
  • 41. Illumina GAII High throughput data analysis Introduction Data storage Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Server network INSERT Total Gb RAM Sequencing pipeline Total CPU Total Tb storage For Genotypin and Database server sequencing services Genotyping applications
  • 42. Summary 1 Classical sequecning method (Sanger) 2 Next-generation sequencing methods Roche 454 ABi SOLiD Illummina GAII 3 High throughput data management 4 High throughput sample preparation 6 Next-NGS sequencing Helicos HeliScope
  • 43. Illumina GAII High throughput sample preparation Introduction Nature Methods, 2010, 7: 111-118 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline High throughput
  • 44. Illumina GAII High throughput sample preparation Introduction Roche Nimblegen RainDance Salid-phase capture with custom- Sample preparation Microdroplet PCR designed oligonucleotide microarray Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Reported 84% of capture efficiency Nature Methods, 2010, 7: 111-118 Reported 65-90% of capture efficiency
  • 45. Illumina GAII High throughput sample preparation Introduction Agilent SureSelect Sample Solution-phase capture with preparation streptavidin-coated magnetic beads Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Reported 60-80% of capture efficiency
  • 46. Summary 1 Classical sequecning method (Sanger) 2 Next-generation sequencing methods Roche 454 ABi SOLiD Illummina GAII 3 High throughput data management 4 High throughput sample preparation 6 Next-NGS sequencing Helicos HeliScope
  • 47. Heliscope Next-NGS: single molecule sequencing Sanger method Nature Reviews genetics, 2010, 11: 31-46 Roche 454 • Any gDNA amplification is requiresd, eliminatign the bias from clonally amplified templates • Low amont of starting gDNA (< 1 μg) ABi SOLiD • More effective quantification in mRNA-seq w/o the amplification step • HeliScope was the first commercialized single molecule sequencer Illumina GAII - Nanopore Poly(A) adaptor linked tot he template fragment One-pass sequencing: poly/T) adaptors are linked to the solid phase
  • 48. Heliscope SBS with reversible terminators Sanger method Nature Reviews genetics, 2010, 11: 31-46 Roche 454 ABi SOLiD Illumina GAII - One colour – real time detection, different form Illumina GAII system Nanopore
  • 49. Nanopore Challenges of Next-NGS sequencing Sanger method Roche 454 ABi SOLiD Illumina GAII HeliScope - Oxford Nanopore: strand-sequencing using ionic current blockage Pacific Biosciences: Real-time DNA sequencing from single polymerase molecules
  • 50. Cosentino Cristian, PhD Genomics and Bioinformatics unit Filarete Foundation – Milan cosentia@gmail.com