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Human Genome CGH Microarray 44B And V2. CGH Protocol Optimization

Human Genome CGH Microarray 44B And V2. CGH Protocol Optimization . Agilent Oligo aCGH is the solution. Provides higher resolution and accurate measurement Great flexibility in probe design and selection that delivers superior performance which meets the demands of your workflow

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Human Genome CGH Microarray 44B And V2. CGH Protocol Optimization

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  1. Human Genome CGH Microarray 44B And V2. CGH Protocol Optimization

  2. Agilent Oligo aCGH is the solution • Provides higher resolution and accurate measurement • Great flexibility in probe design and selection that delivers superior performance which meets the demands of your workflow • Integration of gene expression information for better and faster discovery research via novel data analysis software • Work with whole genome & ng-level input DNA. • Integrated end-to-end solution that supports your entire workflow Oligo aCGH Hybridization and washing Kits NEW NEW Genome-wide Array Custom Array Power of Integration – Infrastructure & Knowledge IMPROVED WORKFLOW (v2.0 Protocol) CGH Application CGH Analytics Software NEW

  3. Human Genome CGH Microarray Kit 44BDesigned for CGH, Validated with samples of known aberrations Designed for genome-wide profiling of genomic aberrations on a single chip. • Genome-wide coverage with average resolution ~35kb • Based on UCSC Genome Browser hg17 (NCBI Build 35) • Probe selection biased toward genes (84% intragenic vs. 16% intergenic) • Optimized probe design for aCGH application • Compatible with current Agilent microarray platform How is different from G4410A ? • Add ~2500 biological probes (New genes from Build 35 and additional gap fillers) • Retain all biological probes from G4410A with exception of 27 probes • Implement CGH eQC grid Part number: G4410B List price: $3750/Kit with volume break Discount: EDU (20%) and VEU Standard catalog array packaging

  4. Probe Design Methodology Goal: Provide a utility tool for high throughput, high resolution genome-wide survey and molecular profiling of genomic aberrations. To construct an array with good coverage of the whole human genome with representation of all known genes emphasizing cancer-related genes and expressed sequences with optimal probe performance.

  5. Probe Design Methodology • Tiled candidate probes across non-repeat masked regions across the genome in 30 bp steps between cut sites for restriction enzymes (RsaI and AluI) • Performed homology search and retained highly specific probes • Learned rules for probe performance dependencies on in silico parameters (thermodynamics, complexity, etc.) using chrX probes and M/F cell-line samples • Filtered homology-searched probe set on in silico parameters • Performed empirical validation on 120,000 semifinal probes using normal M/F cell-line samples and outliers were eliminated • Selected the best set of probes biasing toward genes & exons • Final probe set within a narrow Tm range

  6. Human Genome CGH Microarray Kit 44BDesigned for CGH, Validated with samples of known aberrations • Consisting of ~43,000 60-mer oligonucleotide probes on 1”x3” slide • Genome-wide coverage with average resolution ~35kb* • Content source: UCSC Genome Browser hg17 (NCBI Build 35) • Probe selection biased toward genes 1 probe per well known gene 2 or more probes for each of ~1100 cancer genes Intragenic probes (84%); intergenic probes (16%) • Optimized probe design for CGH application • Ability to detect single copy and homozygous deletions as well as low and high level amplifications • Compatible with current Agilent microarray platform * Calculated by using the total size of the non-repeated genome divided by the total # of features

  7. Genomic Coverage and Probe Distribution • NEW CONTENT FROM PREVIOUS G4410A • 1. Add ~2500 biologicial probes • increase coverage within genomic gaps • cover some missing genes base on Build 35 • 2. Remove 26 probes from 44A • 3. Implement a new eQC grid Chromosome Exonic 16406 Intronic 19805 Intergenic 6685 Position on Chromosome

  8. Genomic Coverage and Probe Distribution Total # of Features 44,290 # of Biological Probes 42,896 # of Unique Biological Probes 42,494 # of Replicated Biological Probes 201 x 3 Un-used Probe Space: 24 Total # of Control probes 1,370 ___________________________________________ Intragenic probes 36,211 (84%) Exonic probes 16,406 (45%) Intronic probes 19,805 (55%) Intergenic probes 6,685 (16%)

  9. Validation using XY/XX model system Two dye-flip pairs of hybridization experiments using 46, XY and 46, XX genomic DNA. 10 pt moving average. * 3ug, direct labeling

  10. Validation using XY/XX model system One dye-flip pair of hybridization experiments using 46, XY and 46, XX genomic DNA. Probability Density * 100ng, amplification Log2 Ratios

  11. Validation using XY/XX model system 100ng Amplification Two dye-flip pairs of hybridization experiments using 46, XY and 46, XX genomic DNA. 10 pt moving average. 3ug Direct

  12. XY vs. XY Experiment (Chr 3)

  13. XY vs. XY Experiment (Chr 17)

  14. Oligo aCGH Protocol (v2.0) Genomic DNA Isolation from Samples Reagent Kits Amplification (100ng) Direct (3ug or more) REPLI-g Kit* Whole Genome Amplification (Phi29) Qiagen Miniprep Kit* Restriction Digestion and Cleanup 7ug 2ug BioPrime Kit, Microcon YM-30* Genomic Labeling, Cleanup Oligo aCGH Hybridization Kit Microarray Hybridization (40 hours, 10rpm) Oligo aCGH Wash Buffer 1, 2 & 3 Wash Procedure B (no wash 3) Wash Procedure A (with ACN, wash 3) OR Scan Ordering Details Data Analysis * 3rd party kits

  15. Oligo aCGH Protocol (v2.0) Customer Benefits Major Improvements Sample Isolation • Quality assessment via E-Gel • Both Direct and Amplified procedures • Hybridization length • Hybridization rotation speed • Wash 3 and No Wash 3 • 44k_CGH_0605 FE protocol • Minimize adverse effects to downstream results due to poor template quality • Minimize any potential bias introduced by amplification, while maintaining the ability to work with small amount of DNA input • Increase signal intensity and robustness • Increase signal intensity and robustness • Operational freedom in standard molecular biology laboratory • More accurate CGH data analysis Sample Preparation Array & Array Processing Data Analysis

  16. Linear increase of BGSubSignal with increasing rotation speed from 5 to 20 RPM (40 h hybs) 20 RPM 10 RPM 20 RPM 5 RPM 10 RPM 5 RPM

  17. Linear increase of Mean_BGSubSignals with increasing hybridization duration (10 RPM) 25 ng XX,XY DNA Repli-G amplification 10 rpm, with Wash 3. Two replicate experiments for each hybridization duration. 17 h 40 h

  18. Linear increase of Median_BGSubSignals with increasing hybridization duration (10 RPM) 25 ng XX,XY DNA Repli-G amplification 10 rpm, with Wash 3. Two replicate experiments for each hybridization duration. 17 h 40 h

  19. ROCArea between 17hr vs. 40hr Slight enhancement in ROC area by increasing hybridization time. 17 h 40 h

  20. Array Images at 17hr vs. 40hr 17hr hybridizations 40hr hybridizations 251270012482 251270012408 251270012417 251270012420

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