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Illumina GAIIx for high throughput sequencing
 

Illumina GAIIx for high throughput sequencing

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    Illumina GAIIx for high throughput sequencing Illumina GAIIx for high throughput sequencing Presentation Transcript

    • Illumina Genome Analyzer IIx for high throughput sequencing Cosentino Cristian, PhD Genomics and Bioinformatics unit Filarete Foundation – Milan (IT) cosentia@gmail.com 15.4.2010
    • Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx Genome Analyzer IIx 3 Data management 4 Target enrichment
    • Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx 3 Data management 4 Target enrichment
    • Approaching to NGS 1 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)
    • Next-generation sequencing platforms 1 Isolation and purification of target DNA Sample preparation Clonally amplified DNAs Single molecule Amplific. Cluster generation Emulsion PCR on solid-phase Chemistry Sequencing by synthesis Sequencing by synthesis with reversible Pyrosequencing Sequencing by ligation with reversible terminators termintors Data analysis Illumina GAII Roche 454 ABi SOLiD Helicos HeliScope
    • Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx 3 Data management 4 Target enrichment
    • Illumina GAII GAIIx instruments 2 Bioanalyzer 2100 Flow Cell Cluster station Genome Analyzer IIx Paired-end module Linux server
    • Illumina GAII Applications 2 de novo sequencing (whole-genome) re-sequencing (whole-genome or targeted) RNA-seq smallRNA-seq CHiP-seq Single-read Paired-end Multiplexing
    • Illumina GAII Sequencing by synthesis with reversible terminator 2 Parameter Performance Amplification Bridge-PCR on solid-phase Chemistry SBS with reversible terminators Cost 2 $/Mbp Advantages Disadvantages •Most widely used platform (> 90 •Low multiplexing capability science/nature publication) •Substitution errors •Sample preparation automatable •SBS, real-time analysis and base calling are performed simultaneously to the run •Automated cluster generation procedure
    • Illumina GAII Coverage estimation 2 Sample 10000 preparation Clusters amplification 1000 Sequencing by synthesis Analysis 35 cycles pipeline 100 50 cycles 75 cycles Coverage/lane 100 cycles 10 35x2 cycles 50x2 cycles 75x2 cycles 1 100x2 cycles 0.001 0.01 0.1 1 10 0.1 Input DNA (Gbp)
    • Illumina GAII GAIIx sequencing workflow 2 Sample Workbench preparation Clusters Cluster Station amplification Sequencing by Genome Analyzer synthesis Analysis pipeline Linux Server
    • Illumina GAII Library preparation 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline gDNA Fragmented Adaptor- Gel purification DNA ligated DNA
    • Illumina GAII Library validation 2 Sample preparation gDNA Clusters amplification Sequencing by synthesis Analysis pipeline smallRNA Total RNA Bioanalyzer 2100
    • Illumina GAII GAIIx sequencing workflow 2 Sample Workbench preparation Clusters Cluster Station amplification Sequencing by Genome Analyzer synthesis Analysis pipeline Linux Server
    • Illumina GAII Flow cell 2 Sample Lanes preparation 1234 5678 Clusters amplification Sequencing by synthesis 120 Analysis tiles/lane pipeline
    • Illumina GAII Cluster generation 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Sample preparation Amplification Cluster Linearization, amplification blocking and primer hybridization SBS sequencing
    • Illumina GAII Bridge amplification 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline 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
    • Illumina GAII Bridge amplification 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Bridge amplification of the reverse fragment Double-strand bridge is formed Double strand bridge is denatured and reverse as wel as forward fragments are covalently attached to the array
    • Illumina GAII Bridge amplification 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Bridge amplification is repeated to enlarge the cluster Double-strand bridges are denatured Reverse strands fragments are cleaved and washed away
    • Illumina GAII Bridge amplification 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Cluster with forward strands only, covalently attached to the array Sequencing primers start the SBS process
    • Illumina GAII Sequencing workflow 2 Sample Workbench preparation Clusters Cluster Station amplification Sequencing by Genome Analyzer synthesis Analysis pipeline Linux Server
    • Illumina GAII SBS technology 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline
    • Illumina GAII GAIIx optical path 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Two colour excitation Four colour emission detection
    • Illumina GAII Single-read sequencing workflow 2 Sample preparation Clusters Cluster amplified amplification FlowCell Sequencing by synthesis Analysis pipeline Install prism Install flow-cell Apply oil First-base incorporation Adjust focus Check quality metrics 36-100 cycles sequencing run for Read 1 Read 1 analysis pipeline
    • Illumina GAII Paired-end sequencing workflow 2 Sample preparation Cluster amplified Clusters FlowCell amplification Sequencing by Install prism synthesis Analysis pipeline Install flow-cell Apply oil First-base incorporation Adjust focus Check quality metrics 36-100 cycles sequencing run Prepare Read 2 for Read 1 36-100 cycles sequencing run for Read 2 Read 1 and 2 analysis pipeline
    • Illumina GAII Paired-end strategy 2 Sample preparation Paired-end sequencing works into GA and uses chemicals from PE Clusters module to perform cluster amplification of the reverse strand amplification Sequencing by synthesis Analysis Single-read pipeline (CS) Paired-end (PEM)
    • Illumina GAII Paired-end strategy 2 Sample preparation Clusters Single-read (read 1) amplification Mapped Unmapped Sequencing by read read synthesis Reference Gene 1 Gene 2 sequence Analysis pipeline Sequence reads Paired-end (read 1 & read 2) Mapped Mapped read read Reference Gene 1 Gene 2 sequence Sequence reads
    • Illumina GAII Sequencing workflow 2 Sample Workbench preparation Clusters Cluster Station amplification Sequencing by Genome Analyzer synthesis Analysis pipeline Linux Server
    • Illumina GAII Firecrest and CASAVA 2 Sample Image files preparation Intensity files Base calls files Clusters amplification Firecrest Bustard From image From intensity Sequencing by synthesis to intensity to reads Analysis pipeline Gerald/ELAND Alignment to genome Assembly Alignment files GenomeStudio Data visualization CASAVA Consensus assembly Sequence ANALYSIS
    • Illumina GAII GenomeStudio viewer 2 Sample preparation Clusters amplification Small RNA sequencing Sequencing by synthesis Analysis pipeline
    • Illumina GAII GenomeStudio viewer 2 Sample preparation Clusters amplification Sequencing by synthesis RNA sequencing Analysis pipeline
    • Illumina GAII GenomeStudio viewer 2 Sample preparation Clusters amplification Sequencing by synthesis DNA sequencing Analysis pipeline
    • Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx 3 Data management 4 Target enrichment
    • High throughput data storage 3 Genotyping units 0.5 – 14 GB/Chip Tape recording unit Data storage for offline backup management server Sequencing unit 250 Tb storage capacity 1 Gbit LAN dedicated 1 – 6 Tb/FlowCell
    • High throughput data analysis 3 External services Sequencing pipeline Database server Genotyping applications Component Availability CPU 40 core equivalent RAM 1 Tb Storage on-line (HD) 250 Tb Storage off-line (Tape) 80 Tb normal 160 Tb compressed
    • Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx 3 Data management 4 Target enrichment
    • High throughput sample preparation 4 Locus lenght or #loci Nature Methods, 2010, 7: 111-118
    • SureSelect target enrichment 4 Agilent SureSelect Solution-phase capture with streptavidin-coated magnetic beads Reported 60-80% of capture efficiency
    • The end Cosentino Cristian, PhD – cosentia@gmail.com