Agilent s CGH Platform: Recent Expansions for Cytogenetic Research

Transcription

1 Agilent s CGH Platform: Recent Expansions for Cytogenetic Research Agilent Microarray and Sequencing Roadshow 2010 Emily Leproust, PhD Director Applications and Chemistry R&D Genomics

2 Table of Contents Introduction Recent Expansions for Cytogenetic Research Expanded probe database Automated sample preparation Microarrays SurePrint G3 CGH microarrays Future: detection of copy neutral aberrations (UPD, cnloh) Literature t review

3 Table of Contents Introduction Recent Expansions for Cytogenetic Research Expanded probe database Automated sample preparation Microarrays SurePrint G3 CGH microarrays Future: detection of copy neutral aberrations (UPD, cnloh) Literature t review

4 Array Comparative Genomic Hybridization = Virtual/Digital/Molecular Karyotyping Virtual karyotypes have ~1000-fold greater resolution than conventional cytogenetics. Agilent is a two-color method, a true comparative genomic hybridization (CGH) platform. Karyotype (Giemsa stain)

5 Aberration associated with Cri-du-Chat Syndrome NA14117, 46,XY,del(5)(qter>p14:).ish del(5) (D5S23-) GEN/CriDuChat%20Website/images/ cri-du-chat_diagram.gif Genome View (Agilent Genomic Workbench) Settings: ADM-2, threshold 10, min 3 probes

6 Aberration associated with Cri-du-Chat Syndrome NA14117, 46,XY,del(5)(qter>p14:).ish del(5) (D5S23-) GEN/CriDuChat%20Website/images/ cri-du-chat_diagram.gif Chromosome View Gene View Settings: ADM-2, threshold 10, min 3 probes

7 Extensions of the Agilent acgh Platform for cytogenetics earray 1. Expanded probe database 3. New microarrays 2. Automated sample prep

8 Table of Contents Introduction Recent Expansions for Cytogenetic Research Expanded probe database Automated sample preparation Microarrays SurePrint G3 CGH microarrays Future: detection of copy neutral aberrations (UPD, cnloh) Literature t review

9 Agilent Offers an Extensive Probe Database for Designing Custom Microarrays for FREE Probe Sequence Database Probe Database containing 28.7 Million computationally validated CGH probe sequences fully available Catalog arrays content incorporates common project needs Customized content incorporates project-specific knowledge or special validation protocols earray: web interface

10 Available on Agilent.com

11 Evolution of the Human CGH High Density (HD) Database million, 400bp spacing Scoring model V1 Probes pass Agilent tm and homology filters One mapping per probe 8 million, 200bp spacing Scoring model V2 Probes pass Agilent tm and homology filters One mapping per probe Used in selection of 244K and 105K CGH catalog products 28.7 million, 40bp spacing Scoring model V3 (handles non unique probes) Probes pass Agilent tm filters One or more mappings per probe (for CNVs and PARs) Used for selection of new 30um catalog designs Probe with >40 perfect matches marked as unmapped

12 Pseudo Autosomal Regions (PAR) PAR1 (2.6 Mb) and PAR2 (320 kb) are homologous sequences of nucleotides on the X and Y chromosomes. PARs allow the X and Y chromosomes to pair and properly segregate during meiosis in males. Aberrations in PARs have been associated with Turner syndrome, Léri- Weill dyschondrosteosis and Madelung's deformity.

13 Coverage of Pseudo-Autosomal Regions SurePrint HD New SurePrint G3 1x244K 2x105K 4x44K 1x1M 2x400K 4x180K 8x60K PAR

14 Table of Contents Introduction Recent Expansions for Cytogenetic Research Expanded probe database Automated sample preparation Microarrays SurePrint G3 CGH microarrays Future: detection of copy neutral aberrations (UPD, cnloh) Literature t review

15 User Manual: Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis Bravo Automated Liquid Handling Platform with Enzymatic and ULS Labeling version 1.0.1

16 Overview of Protocol Steps in blue use the Bravo Automated Liquid Handling Platform.

17 Two Labeling Methods: Enzymatic and ULS Random-primed Klenow fill-in based incorporation of Cy-3 or Cy-5 labeled dntps Universal Linkage System (ULS ) platinumbased direct coupling of Cy-3 or Cy-5 to DNA ENZYMATIC Random primers 10 min Denaturation/Fragmentation 2 hr Klenow Labeling GE Healthcare 96 well DNA Hybridization Wash Scan ULS 10 min Fragmentation 30 min ULS Labeling KreaPURE 96 well + KreaBLOCK

18 Ready-to-go validated protocols, no programming required All required volumes, sample well locations, and tip-usage parameters adjust automatically to account for sample number and array type (1x, 2x, 4x, or 8x).

19 Short Hands-on Time: 60 Minutes for entire CGH workflow for 48 samples To minimize reagent waste the recommended number of arrays processed per Bravo Platform run is: 16, 24, 32, 40 or 48 Bravo Platform can be run multiple times a day

20 Excellent reproducibility 48 replicates of the same sample (500ng Promega Male) and the same reference (500ng Promega Female) was processed on the Bravo Automated Liquid Handling Platform using the enzymatic labeling option 6 Male/Female sample pairs were randomly selected and hybridized on the Agilent SurePrint G3 Human Catalog CGH 4x180K Microarrays (G4449A) The standard deviations of the yields, the specific activity and the microarray QC metrics were very small, indicating the excellent reproducibility of this system In the Male/Female (XY/XX) model system, the log 2 ratios obtained were close to the expected values (-0.96) and there is an excellent separation between autosomal probes and X-chromosome probes indicated by an Area under ROC curve of 97% and a small standard deviation across the samples All QC metrics passed including excellent DLRSD # n=48 * n=6 Metric Average ± Std. Deviation Yield (ug) # 5.4±0.2 Specific Activity Green (pmol/ug) # 17.4±0.7 Specific Activity Red (pmol/ug) # 16.8±0.6 DLRSpread * 0.13±0.003 Signal-to-Noise Green * 87.8±3.8 Signal-to-Noise Red * 125.8±8.8 8 Reproducibility Green * 6.7±1.2 Reproducibility Red * 6.4±1.1 Area Under ROC * 097±0 0.97± Median Diff * -0.96±0.01

21 Comparison with manual processing Labeling method: Enzymatic Microarray format: Agilent SurePrint G3 Catalog CGH 4x180K Number of samples: 10 (5 on Bravo, 5 manual) Cy5 samples: Promega Male (pool of normal males), DiGeorge, Tourette, Prader-Willi, Cri-du-Chat Cy3 samples: Promega Female (pool of normal females)

22 DLRSpread (probe-to-probe log ratio noise) values across 5 different samples sets are equivalent between manual and automated processing DLRSpread 0.25 Good Excellent M-F_Bravo M-F_Manual NA07215_Bravo NA07215_Manual NA10989_Bravo NA10989_Manual NA13555_Bravo NA13555_Manual NA14117_Bravo NA14117_Manual

23 Signal to Noise values across 5 different samples sets are equivalent between manual and automated processing SignalToNoiseGreen i SignalToNoiseRed Excellent Good M-F_Bravo M-F_Manual NA07215_Bravo NA A07215_Manual NA10989_Bravo NA A10989_Manual NA13555_Bravo NA A13555_Manual NA14117_Bravo NA A14117_Manual

24 Array results from the Bravo Platform are identical to the manually processed samples: aberrations associated with DiGeorge Syndrome (NA07215) and Cri-du- Chat (NA14117) were detected with both methods DLRSD DLRSD

25 Summary We have demonstrated an effective new method for automating CGH/CNV sample fragmentation, labeling, purification, and pre-hybridization setup in a 96-well format with the Bravo Automated Liquid Handling Platform The automated workflow showed excellent reproducibility and yielded results that were consistent with manual processing Hands-on time was reduced and throughput was increased for both the standard enzymatic and ULS protocols without compromising data quality Robust, ready-to-go, validated protocols for the CGH workflow, with no setup or programming required

26 Table of Contents Introduction Recent Expansions for Cytogenetic Research Expanded probe database Automated sample preparation Microarrays SurePrint G3 CGH microarrays Future: detection of copy neutral aberrations (UPD, cnloh) Literature t review

27 Flexible Microarrays Designed to Meet Your Specific Needs Agilent s complete CGH/CNV Product Line includes both custom and catalog designs > Agilent microarrays are manufactured under ISO 13485

28 ISO for Arrays and Reagents

29 Agilent SurePrint G3 Human, Mouse and Rat Genome Catalog and Custom CGH Microarrays

30 SurePrint G3 CGH Catalog Designs: Gene Biased Aberrations in or near biologically relevant regions of the genome are of more interest. Genes, mirnas, telomeres, centromeres, Decipher regions.

31 Table of Contents Introduction Recent Expansions for Cytogenetic Research Expanded probe database Automated sample preparation Microarrays SurePrint G3 CGH microarrays Future: detection of copy neutral aberrations (UPD, cnloh) Literature t review

32 Comparison of karyotyping methods Output of Method Method Resol ution Sensitivity UPD cnloh detection Dividing cells needed Distinction of individual clones Screening for new aberrations Balanced translocation Metaphase cytogenetics Low 10% No Yes Yes Yes Yes FISH Low High No No Yes No Yes SNP microarray High High Yes No No Yes No CGH microarray High Very high No No No Yes No UPD: Uniparental Disomy, cnloh: copy neutral Loss of Heterozygosity Adapted from Br J Haematol Sep;146(5): Epub 2009 Jun 26

33 UPD: Uniparental Disomy Both members of a chromosome pair or segments of a chromosome pair are inherited from one parent and neither is inherited from the other parent Uniparental disomy results in an abnormal phenotype when the chromosomes involved are imprinted, such that the genes on these chromosomes are monoallelically active (i.e., only the maternal or paternal allele of the pair is active).

34 Examples of syndromes associated with UPD Prader-Willi syndrome (maternal UPD15) Angelman syndrome (paternal UPD15) Transient neonatal diabetes mellitus (paternal UPD6) Russell-Silver syndrome (maternal UPD7) Beckwith-Wiedeman syndrome (paternal UPD11) MUPD14 syndrome (maternal UPD14) Beckwith-Wiedeman syndrome

35 Augmenting the Agilent CGH microarrays with SNP probes to allow for the detection of copy neutral aberrations Use a subset of probes on a CGH array to measure SNPs in parallel to copy number (on the same array) Workflow unchanged from Agilent Genomic DNA labeling kit: Alu/Rsa restriction digestion / Klenow fragment labeling Addition of SNP content to CGH arrays supplements information from copy number measurements: Parental or sample origin in genetics studies Detection of COPY NEUTRAL aberrations such as Loss of Heterozygosity (LOH) and Uniparental Disomy (UPD) Launch date to be determined

36 Examples: sample associated with Angelman syndrome (left) and Miller-Dieker Lissencephaly syndrome (right) No heterozygous SNPs LOH UPD CGH data SNP data CGH data SNP data

37 Table of Contents Introduction Recent Expansions for Cytogenetic Research Expanded probe database Automated sample preparation Microarrays SurePrint G3 CGH microarrays Future: detection of copy neutral aberrations (UPD, cnloh) Literature t review

38 Curtis et al. (BMC Genomics Dec 09): DNA copy number array technologies assessed Comparison study between Agilent, Affymetrix, Illumina and Nimblegen platforms: assessment of reproducibility, noise and sensitivity Conclusions: Some focal amplifications and deletions were only detected on a subset of the platforms. In particular, Nimblegen failed to detect numerous aberrations that were clear in the other platforms even when probes were tiled in the region of interest. Nimblegen exhibits 2-4fold greater variance amongst replicate probes and variances an order of magnitude greater for replicate array comparisons. In general, for the acgh-based platform Agilent was the best performer and for the SNP-CGH platform, Affymetrix tended to outperform Illumina. Clarity of signal Agilent > Affymetrix > Illumina > Nimblegen

39 Braggio et al. (AACR poster April 10): Side-by-side comparison between Affymetrix SNP6 and Agilent 1M Conclusions: More probe density does not mean more array resolution The side-by-side comparison showed that Agilent 1M array has better exon and gene coverage, lower DLR and higher copy-number detection rate than Affymetrix SNP6.0 array

40 Braggio et al. Higher DLR values in SNP6.0 platform reduces the resolution of abnormality detection, where the SNP6.0 array is unable to call a focal gain on PAX5 identified by 8 and 31 probes using Agilent 244A and Agilent 1M arrays, respectively. In this comparison the Agilent 1M out performed the Affymetrix SNP6. Even with half the number of probes, the 1M was superior in CNA detection, gene/exon coverage and showed less signal variation.

41 Nasri et al. (Cancer Genet Cytogenet Apr 10): Oligonucleotide array outperforms SNP array on FFPE clinical samples

42 Miller et al. ISCA (AJHG May 10) ISCA is a rapidly growing group of clinical cytogenetics and molecular ISCA is a rapidly growing group of clinical cytogenetics and molecular genetics laboratories committed to improving quality of patient care related to genetics laboratories committed to improving quality of patient care related to clinical i l genetic testing ti using new molecular l cytogenetic ti technologies clinical genetic testing using new molecular cytogenetic technologies Total cases to date: 15,751 Total cases to date: 15,751 Pathogenic abnormalities: 2,756 (17.5% Pathogenic of cases) abnormalities: 2,756 (17.5% of cases) Agilent Array designs Agilent Array designs Version 2 July 16, 2009 Version 2 July 16, x 44K: AMADID x 44K: AMADID x 60K: AMADID (popular) 8 x 60K: AMADID (popular) p 2 x 105K: AMADID x 105K: AMADID x 180K: AMADID (popular) 4 x 180K: AMADID (popular)

43 More Information: Available on Agilent.com Product Manuals Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis ULS Labeling for Blood, Cells, Tissues or FFPE Version 3.1 (G ) Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis Enzymatic Labeling for Blood, Cells or Tissues Version (G ) Agilent Oligonucleotidel Array-Based CGH for Genomic DNA Analysis (Bravo Automated t Liquid id Handling Platform with Enzymatic and ULS Labeling) Version (G ) Product Notes Agilent SurePrint G3 Human Catalog CGH Microarrays ( EN) Agilent s SurePrint G3 Human, Mouse and Rat CGH Microarrays ( EN) earray custom CGH microarrays FAQ ( EN) Automating the CGH/CNV Workflow with the Bravo Automated Liquid Handling Platform ( EN) Copy Number Analysis of Archival FFPE Tumor Samples by Oligo Array CGH ( EN)