MeDIP-chip service report

Transcription

1 MeDIP-chip service report Wednesday, 20 August, 2008 Sample source: Cells from University of *** Customer: ****** Organization: University of ***

2 Contents of this service report General information and Summary: customer, sample, array, experiment information and an abstract of the experiment and the data analysis. Data Analysis Results: 1. Peaks_For_CpG_Islands: shows peaks data of the 2 samples which contains all identified peaks overlapping CpG islands. 2. Peaks_For_Genes: shows peaks data of the 2 samples which contains all identified peaks overlapping the promoter region of the relevant transcript (-800bp ~ +200bp). 3. Peaks_For_Genes_Nearest: contains a report listing only the nearest peak to a transcript. 4. Summary_For_CpG_Islands: contains the summary list of peaks overlapping the CpG island for the 2 samples, and genes in the contiguous region of the CpG Island. 5. Summary_For_Genes1: contains the summary list of peaks overlapping the promoter region of the transcript for the 2 samples. 6. Summary_For_Genes2: contains the list of genes in Summary_For_Genes1 which is classified into 3 groups by the criterion whether there is CpG island overlapping the promoter region (-800bp ~ +200 bp) and whether there is at least a peak overlapping the CpG island. DNA QC: 1. Genome DNA Quality Control. 2. Sheared DNA Quality Control. Array Images: contains the array image jpg files. Raw Data: contains signal intensity data extracted from the array images. GFF Files: contains a series of data produced in analysis procedures allowing visualization in SignalMap software. 1. Scaled log2-ratio Data: contains log2-ratio data which is normalized by bi-weight mean algorithm, when ratio is IP vs. Input for individual probes P-value Data: the P-value is positive enrichment score for individual probes. 3. Peaks Data: contains all identified peaks which represent regions of significant positive enrichment. 4. HG18 CpG Promoter Annotation: contains information of primary transcript, tiled regions, CpG islands and TSSs. SignalMap Brief User s Guide (MeDIP).pdf: gives a brief help of the software. FAQ.

3 General information Customer Information Name Institution Department Address Telephone Sample Description Organism: Human Sample Type: cells Sample Number Sample Name Additional Information 1 A 2 B Array information Array type HG18 CpG Promoter Array number 2 Genome build HG18 Hybridizations to be performed Array NO. Cy3 Cy5 Slide1 Input1 MeDIP1 Slide2 Input2 MeDIP2 Experiment Experiment Procedure Status Number of slides 2 Sample Submission Form received OK DNA Isolation OK

4 DNA QC Labeling and hybridization Scanning and data collection OK please see report DNA-QC OK OK Report Example

5 Summary Dear Sir or Madam, We have finished the sample analysis which you submitted. Genomic DNA from cells were extracted according to the manufacturer s instructions and sonicated to random fragments in size about 500bp. Immunoprecipitation of methylated DNA was performed using mouse monoclonal antibody against 5-methylcytidine and Biomag TM magnetic beads coupled anti-mouse IgG. Immunoprecipitated DNA was eluted and purified by phenol chloroform extraction and ethanol precipitation. The Input and IP DNA were labeled with Cy3- and Cy5-labeled random 9-mers, respectively, and hybridized to NimbleGen HG18 CpG Promoter arrays, which is single array design containing all known CpG Islands annotated by UCSC and all well-characterized RefSeq promoter regions (from about -800bp to +200bp of the TSSs) totally covered by ~385, 000 probes. Scanning was performed with the Axon GenePix 4000B microarray scanner. Raw data were extracted as pair files by NimbleScan software. Then, a series of procedures were performed to get the Peaks data with specified parameters (sliding window width: 750bp; mini probes per peak: 2; P-value minimum cutoff: 2; maximum spacing between nearby probes within peak: 500bp). When we get the peaks data, we map it with genomic features: transcripts and CpG islands. The initial map data for each sample are in Peaks_For_CpG_Islands, Peaks_For_Genes and Peaks_For_Genes_Nearest. The summary of the data for 2 samples are in Summary_For_CpG_Islands, Summary_For_Genes1 and Summary_For_Genes2 (More detailed information in Instruction for the Result Data file). NimbleGen s SignalMap data browser enables you to visually interpret the intermediate gff data files with a time limit 30 days. You can download it from NimbleGen s web ( A brief user s guide is available.

6 Below is an example to help you view data (data for reference only). Report Example Maybe, you can select several genes by the methylation situation of your samples, like figure above. Open your peaks data (gff files) and the annotation file in the SignalMap software. Find the proper position,and you can get the picture as below. A CpG island Peaks A Promoter Peaks B CpG island Peaks B Promoter Peaks No Peaks C CpG island Peaks C Promoter Peaks D CpG island Peaks D Promoter Peaks Peaks Please contact us if there is any question.

7 Frequently Asked Questions How do you perform analysis on DNA methylation data? After scaled log2 ratio data is generated a modified ACME algorithm (Method Enzymol. 2006; 411: ) is employed where a fixed-length window is slid along the length of each chromosome, testing at each probe using a one-sided Kolmogorov-Smirnov (KS) test whether the surrounding window is enriched for high-intensity probes relative to the rest of the array. Each probe has a corresponding p-value score (-log10) and a threshold is set to select regions that are enriched (i.e. methylated) in the test sample. Do you normalize DNA methylation data? No, there is no normalization of DNA methylation data. However, we do scale the GFF files by subtracting the bi-weight mean for the log-ratio values from each log-ratio value. This effectively centers the log-ratio values around zero.