Genomic systems 67. Pathologus Kongresszus Keszthely, október 9-11.
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1 Diagnostics Genomic systems 67. Pathologus Kongresszus Keszthely, október Péter Becságh
2 Diagnostics Real Time Cell Analysis RAS Workflow Integration T A C G Cell Analysis Microarrays RT-PCR Sequencing Is a compound effecting a cellular response? Which Gene expression pathways are altered by the compound effect? What is the quantitative effect of the compound on relative gene expression? Is a certain genomic pattern associated with the effect?
3 System Solutions
4 Introduction Magnetic Bead Technology
5 Introduction MagNA Pure LC System - Fully automated benchtop system can purify up to 32 samples and setup PCR into different vial formats - Innovative, reliable Magnetic Bead Technology - Highly flexible - purifies nucleic acids from almost any sample using appropriate kits - User-friendly software protocols are optimized to meet your experimental needs
6 Introduction MagNA Pure Compact System - Small footprint instrument automatically processes 1 8 samples in 22 to 30 minutes. - Ready-to-use reagents and disposables are convenient, safe and allow easy run setup. - Streamlined software is easily navigated with a touch-screen and can receive data input from a barcode scanner. - Time-saving, state-of-the-art user management system. - Host connectivity to simplify data transfer and storage.
7 Principle of the PCR Method r Separation of the nucleic acid double strand (DNA) 2. Annealing of short DNA-fragments (Primers) on their specific sequences 3. Elongation (de novo synthesis) of these short fragments by Taq-Polymerase 4. Detection by specific probes 4.
8 The LightCycler System Accurate. Reproducible. Reliable. LightCycler 1.5 Instrument Proven performance that anyone can afford LightCycler 2.0 Instrument Enhanced performance that meets the needs of even the most demanding user
9 LightCycler 480 System Automated Plate Loading E.g., Twister II Plate Handler (Caliper Life Science)
10 Patient Info LIS File/Data/Setup/Barcode Sample Prep Amp & Detect Retrieve Software Sample ID, Test ID Sample ID, Result manual transfer Sample & Reagents X480 Sample Prep Station Z480 Thermal Cycler Report
11 Diagnostics Gene Scanning High-Resolution Melting Analysis Example: Sequence variations (SNP G T) in the LPLH3 gene 72 samples, 164 bp amplicon heterozygous (homo and heteroduplexes) homozygous wildtype (homoduplexes) homozygous mutant (homoduplexes)
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13 Scorpion technology
14 Diagnostics
15 DxS EGFR mutációs tesztkészlet: az epidermális növekedési faktor receptor (EGFR) gén 29 mutációjának kimutatására Diagnostics
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17 Genome Sequencer FLX titanium System
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20 Key Concept 454 Sequencing from individual DNA molecules Library of single stranded DNA molecules One DNA molecule per bead Clonal amplification of that single molecule to ~10 million copies Independent sequencing of each bead One Bead = One Read = One DNA molecule
21 Sequencing Workflow Short hands-on time, quick total time to result empcr Prep Run Sequencing Hands-on Time Total Time 4 h 8 h 2 h 2 h 0 h 7 h Total Hands on Time Total Time 6 h 17 h
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23 GS FLX System Flexibility Multiplex Identifiers (MIDs) Sequencin g primer Primer A Read Key MID Library fragment Primer B MID1 MID2 MID3 MID4 MID5 MID6 MID7 MID8 MID9 MID10 MID11 MID12 ACGAGTGCGT ACGCTCGACA AGACGCACTC AGCACTGTAG ATCAGACACG ATATCGCGAG CGTGTCTCTA CTCGCGTGTC TAGTATCAGC TCTCTATGCG TGATACGTCT TACTGAGCTA T A C G T A C G T A C G T A C G T A C G The MIDs in flow space
24 GS FLX System Flexibility Multiplex Identifiers (MIDs)
25 Workflow for SeqCap & 454-Sequencing Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Fragment and hybridize to Nimblegen capture array Elute gdn A 454 Sequencing Analyze Exon Sequences
26 Medical Research based on GS Amplicon Re- Sequencing High confidence exon resequencing (e.g. heterozygote calling) Sensitive detection of somatic mutations in cancer specimens SNP screening in pooled samples Sensitive detection of low frequency resistance mutations in viruses
27 Cancer Exon Sequencing Sequence Reads Replicate 1 Replicate 2 Replicate 3 Probes Target Exons 6732 cancer exons captured 5 Mb base of total sequence (0.15% of the genome) Three replicate capture/sequencing experiments Albert TJ, et al. Nat Methods (11):903-5.
28 Example: Low Tumor Content Mutation Detection in Pre-therapy NSCLC Sample (somatic mutations) Matthew Meyerson, Dana Farber Institute and 454 Life Sciences EGFR exons from paraffin-embedded pleural effusion material from a NSCLC patient (patient 12.3) who initially responded strongly to erlotinib treatment Sequencing revealed an 18 base pair deletion in exon 19 at a frequency of 0.28% that might underlie the patients early strong TKI response.
29 Mutation Detection in Relapse NSCLC Sample - Low Tumor Content Matthew Meyerson, Dana Farber Institute and 454 Life Sciences Following 12.5 months of erlotinib treatment patient 12.3 relapsed. Del-4 mutation at 3% abundance Exon 19) In addition, T790M mutation, at 2% (Exon 20) shown in previous studies to confer resistance to TKI inhibitors
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31 MANY THANKS!
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