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the agilent Microarray platform and its applications
- 1. The Agilent Microarray Platform MrDhananjay Pathak Senior analyst Metropolis Healthcare limited GRL Mumbai. An array of possibilities
- 2. Agilent Microarray Platform Hardware V ForResearch Use Only. Not for use in diagnostic procedures. Reagents Slides You • Scanner • Hybridization oven • Hyb chambers • Automation • Dedicated labelling kit • Hybridization kit • Quality controls • Washing buffers • Microarray slides for different applications / species • Multiple array formats • Unlimited customization capabilities • No minimum order • No extra cost • Same quality as catalog arrays
- 3. 1M 400K 180K 60K Agilent Microarray FormatsTimeline 244K 44K 105K 15K 2005 2006 2007 2008 Existing Most common 22K 44K 1.9K 11K Multiple x 2001 2002 2003 2004 Obsolete V For Research Use Only. Not for use in diagnostic procedures.
- 4. 2000 22K 135 m 2006 244K 55 m All images1 sq mm Evolution of Ink-Jet Oligo Array Technology 2008 1000K 30 m 2004 44K 100 m General scanning resolution rule... Resolution=spot diameter/10 This ensure to have enough informative pixels for each feature V For Research Use Only. Not for use in diagnostic procedures.
- 5. Agilent Microarray Formats SurePrintG3 formats include approximately 4 times more content and requires high resolution scanning (3 µm) G3 format include complete CGH+SNP and CGH Product Line formats for catalog and custom Increased content decreased cost/sample Agilent SurePrint HD Agilent SurePrint G3 V For Research Use Only. Not for use in diagnostic procedures.
- 6. Microarray technology Oligo synthesis ForResearch Use Only. Not for use in diagnostic Agilent’s inkjet process has a 99.5% per-cycle yield with no depurination, producing the most full-length products at any oligonucleotide length. Why Cycle Yield is important: Cycle yield loss results from coupling and deblocking failures. • Coupling failures cause truncated sequences. • Deblock failures (irrecoverable) cause random deletions • Depurination creates truncated sequences Oligo length and quality influences: Specificity of hybridization Reproducibility Signal to noise ratio Y
- 7. Binding events arecooperative! Any error, even one, in a probe sequence will greatly reduce the signal-to-noise ratio because the equilibrium of binding/not binding is severely tilted towards not binding. Error-free probes and target hybridization, equilibrium One error in the probes (on average), imbalanced equilibrium Two errors in the probes (on average), imbalanced equilibrium Probe Target Probe Target * * * * * * * * Signal intensity = 1000 Signal intensity = 200 Signal intensity = 100 * * * * * * * Probe Target Better sensitivity Increased specificity Wider dynamic range Accurate copy number And transcript detection Microarray technology Importance of oligo quality Y For Research Use Only. Not for use in diagnostic procedures.
- 8. While number ofprobes on the array has just limited impact, probe length really makes the difference • Longer probe gives higher specificity → ability to detect with precision also complex r e g i o n s • Less probes are needed to confidently make a CN call or to detect an expressed gene → so less probe are needed to achieve s a m e performances • Longer probe provide better sensitivity → ability to work also with complex/lower q u a l i t y samples • Increase specificity and sensitivity provide a wider dynamic range → enabling better CN call, more precise profiling Microarray Technology Importance of oligo lengh Y For Research Use Only. Not for use in diagnostic
- 9. A T AT C G C G A T C G T C G A T C A T C G A T G A T C G A C G A T C G T C G A T C Microarray Technology How they are made Data provided to the printer for array production is a simple matrix This allows Agilent to provide unlimited customization capabilities, assuring catalog grade quality with no additional cost. Specific hydrophobic characteristics of the glass associated with inkjet printing technology create microdoplets enabling synthesis to be carried out in an optimized environment. This results in: • High efficiency • Low error rate • No truncated oligos Y For Research Use Only. Not for use in diagnostic
- 10. In-house serial dilutiondata App Note 5990- 9129EN Probe Quality (Length & Accuracy) & 2 color approach allow a better copy number calls important for mosaicism detection and clonal fraction determination Agilent ability to call with high accuracy copy number allow to detect mosaicism without need to use SNP data (Affymetrix needs SNP to supply poor CN call accuracy) Low level mosaicism can be picked up (down to 8%, Valli et al. Molecular Cytogenetics 2011, 4:13) More forgiving with regards to DNA quality. Agilent data quality is very high with good quality samples; poorer quality samples (e.g. FFPE) will still give good results Y For Research Use Only. Not for use in diagnostic Microarray technology Important technical highlights: oligo length and quality
- 11. Microarray technology Important technicalhighlights: 1 vs 2 color approach RELEVANT FOR: 1 COLOR : no need to run reference with sample, less sensitive to detect small changes in CN (for example 3 vs 4 copy), sensitive to reference selected for analysis 2 COLOR: reference run with every sample, takes into account experimental variability, easier to detect also small changes in CN, leading to more reliable CN call Comparative Genomics Hybridization Gene Expressio n Y For Research Use Only. Not for use in diagnostic
- 12. For Research UseOnly. Not for use in diagnostic procedures. Data Interpretation - CGH Agilent is the only company that offers both data analysis and interpretation capabilities. Alissa Interpret (part of Alissa Clinical Informatics Platform) can analyze CGH data and integrate them with NGS Combined CNV+NGS variant triaging and evidence-based curation drives sample to answer for clinicians Alissa Interpret isa USA Class I Exempt M edical Device, Europe CE IVD, Canada and Australia Class I IVD Device Y
- 13. For Research UseOnly. Not for use in diagnostic procedures. Example workflow: CGH Prenatal Research Postnatal Research Cancer PGT • Follow up after NIPT screening • Screening for trisomies and micro-dup/deletions • Genome-wide screening for DD/ID/CA/ASD • Confirmatory or reflex test to NGS • Genome-wide screening CNV+LOH in solid and heme-onc tumors • Exon-level analysis of cancer-specific genes • Aneuploidy screening to rank embryos • Segmental dup/del analysis EasyChip - customized prenatal assay New GenetiSure Cyto Microarrays GenetiSure Cancer Research Arrays/Baylor Cancer Arrays GenetiSure Pre- Screen Arrays SureScan Microarray Scanner CytoGenomics & Alissa Y
- 14. Editing will introduce modification in genome needto evaluat e editing effects Need to identify possible secondary insertion sites For Research Use Only. Not for use in diagnostic procedures. Example workflow: GE Genome editing Cancer Clinical Research Biomarker Research Screen biomarkers in specific disease Identify disease specific signature Develop drugs/ test adressing identified biomarker miRNA are excellent biormarkers, often showing changes at early stage of disease. They are present in plasma and serum, and in EVs (exosomes) Classify different tumor types Identify cancer specific pathways Follow up dissease progression Microarrays provide fully validated protocols from sample to data analysis. Agilent GE MA allow direct comparison of normal and cancer tissue, enabling an accurate analyis in less than 2 days starting from limited amount of sample. GE Microarrays are a perfect fit as follow up of editing experiments: Accurate profiling cheap, highthrouput and fast Possibility to customize for dedicated pathways Increased ability to detect small changes in low expressed genes respect to RNAseq V
- 15. For Research UseOnly. Not for use in diagnostic procedures.