High-Resolution Quantitative Methylation Profiling with EpiTYPER and the MassARRAY System

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1 High-Resoltion Qantitative Methylation Profiling with EpiTYPER and the MassARRAY System A complete system for discriminating methylated vs. non-methylated DNA; incldes software, reagents, and MALDI-TOF mass spectrometry for detection and qantification. Interrogate 10s-100s of samples and CpG sites in amplicons from bp and detect down to 5% differences in methylation. Validate methylation array, next gen seqencing, or gene promoter stdy reslts. Referenced in over 400 pblications in diverse fields, inclding cancer, differentiation and development, diagnostics research, metabolism, and epigenetics. For Research Use Only. Not for se in diagnostic procedres.

2 Overview Agena Bioscience's DNA methylation analysis technology (EpiTYPER) is one of the most reliable qantitative methods available today for analyzing DNA methylation changes 1,2. The technology, which has been referenced in more than 400 peer-reviewed jornal articles, incldes the following components: EpiDesigner - software for genomic target selection and PCR primer design. EpiTYPER Reagent Sets- reagents and consmables for all downstream processes, following bislfite treatment of DNA. MassARRAY Analyzer 4- MALDI-TOF mass spectrometer for robst and precise signal detection and qantification. EpiTYPER Reporting Software- for data analysis and graphical presentation of the level of methylation at each CpG site in each sample. Benefits of the EpiTYPER DNA Methylation Analysis Technology EFFICIENCY Go from bislfite-treated DNA to data in 8 hors. Covers mltiple CpGs in amplicons of p to 600 bp. Compatible with many sample types, inclding formalin-fixed paraffin-embedded tisse. PRECISE & CURATE High precision (5% CV). High inter-laboratory reprodcibility. SENSITIVE Detects down to 5% change in methylation levels. COST EFFECTIVE 24-, 96-, and 384-well formats available. Mltiple CpGs analyzed in one simple reaction, from amplicons as long as bp. SIMPLE WORKFLOW No need to design CpG-specific primers. No PCR prodct prification needed. Ideal for investigating a few or several hndred target regions. Convenient software soltions for comparison between samples. How it Works EpiTYPER biochemistry starts with bislfite treatment of genomic DNA, followed by PCR amplification of target regions. The reverse primers contain a T7 promoter tag. Next, in vitro RNA transcription is performed, followed by base-specific RNA cleavage. Finally, the cleavage prodcts are analyzed sing MALDI-TOF mass spectrometry (MassARRAY Analyzer). The methylated and non-methylated cytosine resides in the original genomic DNA are easily distingished sing EpiTYPER Software. For Research Use Only. Not for se in diagnostic procedres.

3 Workflow - From Assay Design to Reslts STEP 1: ASSAY DESIGN EpiDesigner is an online atomated design tool for DNA methylation experiments on the MassARRAY System. Jst enter yor target seqences and the software determines primer designs for the most complete DNA coverage. In addition to optimized primer seqences, EpiDesigner delivers an easy-to-read graphical interpretation of the amplicons designed over yor target regions, as well as annotating distinct CpG sites covered by the assays. STEP 2: BISULFITE TREATMENT Genomic DNA from all samples is extracted and treated with bislfite. This treatment converts any non-methylated cytosine resides into racil (shown below in red), while methylated cytosine resides (ble) are naffected. This step reslts in the generation of methylation-dependent seqence changes in the DNA template. STEP 3: PCR, IN VITRO TRANSCRIPTION, AND RNA CLEAVAGE DESIGN The EpiTYPER Assay starts with PCR sing T7-promotertagged reverse primers to amplify the target regions while preserving the bislfite-indced seqence changes. After SAP treatment, in vitro transcription is performed and the reslting RNA transcripts are specifically cleaved at racil resides. The reslting fragments differ in size and mass, depending on the seqence changes generated throgh bislfite treatment. This difference allows the data analysis software to generate qantitative information for each analyzed target fragment. STEP 4: DATA QUISITION AND ANALYSIS The EpiTYPER reaction prodcts are dispensed onto a SpectroCHIP Array (Chip). The Chip is then placed in the MALDI-TOF mass spectrometer for data acqisition, which typically reqires mintes. The reslts are atomatically loaded into a database for data analysis with EpiTYPER software. Figre 1. Overview of EpiTYPER Assay. Ncleotides that determine U-specific cleavage are shown in lower-case for illstration prposes. NON-METHYLATED GENOMIC DNA METHYLATED GENOMIC DNA Genomic DNA a t a a a t t t a t a a a t t t Bislfite-converted forward strand UGa UG UG a UG aug a a a a a PCR In vitro transcription TGa t TG TG a t TG atg t a t T7 a t a t a t a t T7 RNA RNA fragments after racil-specific cleavage #1 #2 #3 #1 #2 #3 MALDI-TOF MA analysis prodces signal pattern pairs indicating non-methylated (red) and methylated DNA (ble) Intensity #3 #1 #2

4 EpiTYPER Software The EpiTYPER software provides an advanced and convenient soltion for the qantitative analysis of CpG methylation. Nmerical and graphic interpretation tools are available and the data are atomatically matched to the provided seqence. Basic statistical analysis and confidence ratings are available for bilt-in qality control. DATA ANALYZER MODE Data Set Selection Clicking on one or both of these bttons will determine which data set is inclded in tab panes and EpiGram. Spectrm Pane Atomatically displays the mass spectrm for all analyzed amplicons and identifies the selected CpG site (red arrow in spectrm). Methylation Pane Obtain cstomized visal presentation of amplicon data in the most sefl format for yor needs. Seqence View Pane This slender display shows the entire forward and reverse ncleotide seqence for the selected amplicon. CUSTOM VIEWS OF AMPLICONS Display Options These mens allow fine-tning of program parameters, panes to display, and methylation color coding of CpG sites. Epigram Pane This pane provides graphical representations of the CpG sites within the selected amplicon. Each is color coded to represent the degree of methylation, providing a qick, reliable comparison between samples and CpG sites.

5 MassARRAY System An Ideal Genetic Analysis System EpiTYPER DNA methylation analysis is performed on Agena Bioscience's MassARRAY System, which ses MALDI-TOF mass spectrometry for robst and precise signal detection and qantification. The MassARRAY System can also be sed for other ncleic acid analysis methods, inclding SNP and somatic mtation detection, and indel and CNV analysis. All methods offer flexible assay design, fast timeto-reslts, and the ability to rn from tens to thosands of samples daily. These featres make it the ideal genetic analysis system for validation and fine mapping stdies in basic and translational research settings. The Complete Soltion for Genomic Analysis HIGH PERFORMANCE Accrate MALDI-TOF mass spectrometry detection provides nparalleled specificity and sensitivity for the most reliable reslts. MAXIMUM FLEXIBILITY Analyze any combination of SNPs and samples to meet varying stdy reqirements. HIGHLY SCALABLE 24-, 96-, and 384-well options for low to high-throghpt applications. EASY TO USE Data acqisition software streamlines yor workflow, and robst assay design software atomates primer design and optimization to maximize efficiency and minimize experimental variability. A VARIETY OF GENOMIC APPLICATIONS Ready-to-se reagent sets for somatic mtation analysis, genotyping, methylation analysis, and qantitative applications (gene expression and copy nmber variation) provide a broad and flexible men with short trnarond time. CHALLENGING SAMPLE TYPES De to the short amplicon lengths needed for the assays, virtally all DNA sample types are amenable to analysis on the MassARRAY System. Blood plasma Serm Fresh frozen tisse Biopsy samples Formalin-fixed tisse samples Micro-dissected cells Bccal cells Ear pnches Hair follicles

6 Ordering Information Reagent sets are available in 24-, 96-, and 384-well formats, and are designed for se with the MassARRAY System with EpiTYPER software. EpiTYPER COMPLETE REAGENT SETS EpiTYPER CESSORY REAGENTS CAT. NO. DESCRIPTION FORMAT CAT. NO. DESCRIPTION FORMAT Complete EpiTYPER Methylation Reagent Set Reagents, SpectroCHIPs, and PCR 10219A MassCLEAVE T7 Kit (T Cleavage) Reagents only Complete EpiTYPER Methylation Reagent Set EpiTYPER Reagent and SpectroCHIP Set EpiTYPER Reagent and SpectroCHIP Set 8 x 24 Reagents, SpectroCHIPS, and PCR Reagents and SpectroCHIPs 2 x 384 Reagents and SpectroCHIPs PCR Accessory and Enzyme Set (3,840 reactions) PCR Accessory Set (3,840 reactions) PCR Enzyme (3,840 reactions) EZ 96 DNA Bislfite Treatment Kit PCR only PCR only PCR only 192 Rxns Bislfite kit EpiTYPER Reagent and SpectroCHIP Set 2 x 96 Reagents and SpectroCHIPs EZ DNA Bislfite Treatment Kit 50 Rxns Bislfite kit Pblished Stdies Using the MassARRAY System Visit to search or online database for pblished stdies sing the MassARRAY System in yor area of interest. REFERENCE 1. A systematic comparison of qantitative high-resoltion DNA methylation analysis and methylation-specific PCR. Clas R, et al. Epigenetics 7(2012) Qantitative high-throghpt analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Ehrich M, et al. PNAS 102(2005) Agena Bioscience, Inc General Atomics Cort San Diego, CA Phone: Order Desk: Order Desk Fax: Order Desk orderdesk@agenabio.com Web: agenabioscience.com US GENOME EU AP JP CN For Research Use Only. Not for se in diagnostic procedres. MassARRAY, EpiTYPER, and SpectroCHIP are registered trademarks of Agena Bioscience, Inc. Agena Bioscience is a trademark of Agena Bioscience, Inc Agena Bioscience, Inc. All rights reserved R