 |
|
Roche NimbleGen offers five types of promoter designs to suit your DNA Methylation analyses: 2.1M Deluxe Promoter Arrays, 385K Two-Array Sets, 385K RefSeq Promoters, 385K RefSeq XM Promoters, and 385K Minimal Promoters.
Compare Promoter Designs
Roche NimbleGen offers five types of promoter designs to suit your DNA Methylation analyses:
- 2.1M Deluxe Promoter Arrays are single array designs for human and mouse that cover ~10 kb of all annotated promoters. In addition, these arrays tile through all annotated CpG islands, all annotated miRNA promoters, and manually selected ENCODE regions (human only). All regions are tiled at 100bp probe spacing. These arrays also include new positive, negative and non-CpG control regions to facilitate assessment of array and sample performance. NEW!
- 385K Two-Array Promoter Sets contain all annotated splice variants and alternative transcription start sites. This array set provides the most comprehensive tool for genome-wide mapping of transcriptional regulatory elements including all RefSeq genes, the Mammalian Gene Collection, and UCSC known genes to encompass the most comprehensive list of transcripts available. Designs are available for human, mouse, rat, and Arabidopsis thaliana.
- 385K RefSeq Promoter Arrays are single array designs containing all known well-characterized RefSeq genes (NM prefix). The promoter regions on these arrays are covered by 50-75mer probes with approximately 100bp spacing, dependent on the sequence composition of the region. Designs are available for human, mouse, and rat.
- 385K RefSeq XM Promoter Arrays are single array designs containing model reference sequences produced by NCBI's Genome Annotation Project (those accessions beginning with XM) that are predicted by genome sequence analysis. The entries represent ab initio predictions, or have some level of transcript or homology to known genes to support the gene predictions. They represent the transcripts and proteins that are annotated on the NCBI Contigs, and they may be different from GenBank submissions for mRNAs and/or the curated RefSeq records with NM, NR, NP accession prefixes. Designs are available for human and mouse.
- 385K Minimal Promoter Arrays are currently available for Arabidopsis thaliana.
385K Promoter Design Specs
| | Design Type | Design Name | Regions | Avg. Region Size | Total Coverage | Transcripts Represented |
A
R
A
B
I
D
O
P
S
I
S | Two-Array Set | ATH6 promoter 1 of 2 | 6335 | 6334 | 40Mbp | 15177 |
| Two-Array Set | ATH6 promoter 2 of 2 | 6442 | 6321 | 41Mbp | 15392 |
| Minimal Promoter | ATH6 min promoter | 24173 | 1681 | 41Mbp | 30516 |
H
U
M
A
N | RefSeq Promoter | HG18 RefSeq promoter | 18028 | 2819 | 51 Mbp | 24659 |
| Two-Array Set | HG18 promoter 1 of 2 | 11417 | 4745 | 54 Mbp | 29028 |
| Two-Array Set | HG18 promoter 2 of 2 | 11630 | 4770 | 56 Mbp | 30329 |
| RefSeq XM Promoter | HG18 RefSeqXM promoter | 4910 | 11917 | 59 Mbp | 8045 |
M
O
U
S
E | RefSeq Promoter | MM8 RefSeq promoter | 17354 | 2582 | 45 Mbp | 19489 |
| Two-Array Set | MM8 promoter 1 of 2 | 10807 | 4422 | 48 Mbp | 24096 |
| Two-Array Set | MM8 promoter 2 of 2 | 10825 | 4412 | 48 Mbp | 24209 |
| RefSeq XM Promoter | MM8 RefSeqXM promoter | 5980 | 9496 | 57 Mbp | 8136 |
R
A
T | RefSeq Promoter | RN34 promoter | 15398 | 2873 | 44Mbp | 21632 |
| Two-Array Set | RN34 promoter 1 of 2 | 7877 | 6050 | 48Mbp | 11280 |
| Two-Array Set | RN34 promoter 2 of 2 | 8034 | 6073 | 49Mbp | 11553 |
To download a spreadsheet of all the genes whose promoters are tiled on NimbleGen 385K promoter arrays, click here for HG18 or here for MM8. The start column indicates the starting position of the transcript in the genome; if the transcript is on forward (+) strand, this is the putative transcription start site. The stop column indicates the ending position of the transcript in the genome; if the transcript is on reverse (-) strand, this is the putative transcription start site.
Literature
For a complete listing of literature covering all Roche NimbleGen products and services please visit our literature page.
FAQ
| Hide All Topics Show All Topics |
| Sample Preparation |
| What kind of samples can be used on NimbleGen's DNA methylation arrays? |
These arrays are designed to detect differences between a sample enriched for methylated DNA and a control sample, such as total genomic DNA. We recommend samples enriched using affinity-based methods that utilize an anti-5-mC antibody or MBD proteins. We also accept enzyme based methods to detect DNA methylation, such as Hpa II tiny fragment enrichment by ligation-mediated PCR (HELP). |
| Do you have a recommended protocol for front-end sample processing for producing DNA fragments enriched for methylation? |
Yes, we recommend the MeDIP (methylated DNA immunoprecipitation) protocol (Nat Genet. 2005 Aug;37(8):853-62) followed by amplification if necessary. Please contact NimbleGen's Technical Services for a detailed sample preparation protocol. |
| What is the minimum amount of DNA required to perform a NimbleGen recommended MeDIP experiment? |
We recommend starting with 5μg high-quality genomic DNA. However, even smaller amount DNA can be used (1μg or even 200ng). The IP process generally yields 5-10% of the original starting DNA, and you can amplify your IP-ed DNA using whole genome amplification (WGA; kit available from Sigma) in order to obtain at least 4μg of DNA. |
| Do I need to amplify my MeDIP samples? |
We are able to use unamplified samples for labeling reactions if at least 2.5μg of enriched methylated DNA was obtained. Greater amounts of enriched DNA can be obtained by starting with more DNA and by pooling samples from multiple experiments. If amplifcation is necessary, we recommend using the WGA kit (Sigma, catalog #WGA2-50RXN) for MeDIP samples. We have routinely observed that the WGA method introduces little bias during the amplification. |
| What is the effect of CpG content on the MeDIP reaction? |
Studies by Weber et al. (Nat. Genet. 2005, 37(8):853-862) have shown that the more more methylated CpG dinucleotides within a given fragment, the more DNA that is immunoprecipitated. The increase immunoprecipitated material is due to the 5-methylcytidine antibody having more available epitopes (methylated CpG). |
| What are the sample requirements for DNA methylation arrays? |
We require 4μg DNA (although 2.5μg is acceptable) at a concentration of 250-500ng/μl with the majority of fragments greater than 200bp. The A260/A280 ratio should be at least 1.7 and the A260/A230 ratio should be at least 1.6. |
| Does NimbleGen perform front-end sample processing (e.g. IP and amplification)? |
No, NimbleGen is not currently set up to perform contract DNA methylation experiments for our customers. |
| Should I use a negative control and if so what should I use? |
Many of our customers do not use a negative control (e.g. nonspecific IgG antibody), but it is recommended if this is your first experiment with NimbleGen. You will want to have your negative control (IgG) co-hybridized with total DNA (input) in order to avoid a high signal/noise ratio that is often seen when the IgG sample is co-hybridized with the immunoprecipitated sample. |
| What should I use as a reference sample? |
The majority of our customers use total (input) sample as a reference. Using a nonspecific IgG is not a suitable reference. |
|
| Array Design |
| Do we have a catalog CpG island array? |
Yes, we now offer our Human and Mouse 2.1M Deluxe Promoter Arrays for that include all annotated CpG islands and extensive coverage of all annotated gene and miRNA promoters. These arrays also include new positive, negative and non-CpG control regions to aid in assessment of experimental performance. (continue with previous info starting with) We also offer human, mouse, and rat CpG Island Plus Promoter arrays that include all UCSC annotated CpG islands plus 1kb human promoter or 1.8kb mouse promoter region of every RefSeq gene. Control tiled regions on the array include the HoxA cluster (methylation hotspot) and several imprinted loci, including H19/IGF2, KCNQ1, and IGF2R. |
| Are the probes designed from both strands? |
No, NimbleGen only designs probes based off of the forward strand. |
| How does NimbleGen address repetitive elements in the genome for DNA methylation designs? |
We have developed our own method of repeat masking which is dependent on the mean frequency of the 15-mers which make up each oligo. A table is made of the count of all 15-mers that appear in the genome, from both strands. Then a 15-mer window is slid along each oligo, looking up the count of each 15-mer in the table, and calculating the average count. A threshold is set, usually 100 for large eukaryotic genomes, and any probe that exceeds that threshold is eliminated from further consideration. Depending on the region of the genome being evaluated, approximately 20-25% of the DNA is excluded. For some designs we use conventional repeat masking, as done by the RepeatMasker program http://www.repeatmasker.org/). However, NimbleGen has no access to the repeat libraries necessary to use this application, so we rely on third parties to supply this type of masked sequence. We find, however, that RepeatMasker is often overly aggressive and can mask 50-55% of human DNA sequence. See the following paper for reference: Bioinformatics. 2006 Jan 15;22(2):134-41. WindowMasker: window-based masker for sequenced genomes; Morgulis A, Gertz EM, Schaffer AA, Agarwala R; National Center for Biotechnology Information, National Institutes of Health, Department of Health and Human Services Building 38A, Room 1003N, 8600 Rockville Pike, Bethesda, MD 20894, USA. |
| What spacing do you recommend for DNA methylation designs? |
We recommend probe spacing of 100bp or less. |
| Can I get a graphical representation of all probes for a cataloged design that I am interested in so I can see what regions of the genome have coverage? |
Yes, we can generate this information in GFF format for all of catalog designs. You will need a copy of Roche NimbleGen's SignalMap software to view the GFF files. A free, 30-day demo version of SignalMap is available for download. |
|
| Data Analysis |
| Does NimbleGen 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. |
| How does NimbleGen perform analysis on DNA methylation data? |
After scaled log2 ratio data is generated a modified ACME algorithm (Method Enzymol. 2006; 411:270-282) 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. If you are a DNA Methylation array customer and are stil using NimbleScan 2.3 you will need to download the ACME plug-in by clicking here. |
| Why is the data analysis for DNA methylation different from ChIP-chip? |
Since DNA methylation array data is often characterized by broad ranges of enrichment (whereas ChIP-chip data enrichment is more discrete) a different method to identify peaks from scaled log2-ratio data must be employed. |
| How do I validate a genome-wide DNA methylation array experiment? |
Common methods include bisulfite sequencing (single nucleotide resolution) to validate methylation status of all CpG dinucleotides within a peak called on the array, combined bisulfite restriction analysis (COBRA, determine percent methylation), and quantitative PCR to validate MeDIP array results by accurately measuring enrichment of methylated DNA fragments. |
|
| Deliverables |
| Can I get the images for my data? |
Yes, we can supply you with the raw data array images (.tif) upon request. Scaled log2-ratio data (.gff) files and peak (.gff) files are included in your deliverable data. |
| Can I get a graphical representation of all probes for a cataloged design that I am intersted in so I can see what regions of the genome have coverage? |
Yes, we can generate this information in GFF format for all of the catalog designs. You will need a copy of Roche NimbleGen's SignalMap software to view the GFF files. A free, 30-day demo version of SignalMap is available for download. |
| Does NimbleGen generate reports listing the most significant binding/modification events for my DNA methylation experiment? |
Yes, we are including two summary reports that map the peaks from your DNA methylation data relative to the transcription start site of a gene. For instance, if a peak is called within the promoter region of a gene, the report will give the approximate location of the peak as a negative position (upstream of the start site) or positive position (downstream). Also included are accession number of the gene, gene ID, chromosome position, among others. These reports will help narrow down the genomic regions you should be looking at when moving forward to validate your DNA methylation data (e.g. bisulfite sequencing or qPCR). |
|
| Benefits |
| Why should I use a genome-wide approach to study DNA methylation? |
NimbleGen's DNA methylation analysis arrays allow you to map DNA methylation across your genome of interest genome in an unbiased fashion, compare differential methylation patterns between cells, tissues, and tumors, and identify prognostic markers. |
| How sensitive are NimbleGen's DNA methylation arrays? |
Our arrays, when coupled with MeDIP, can readily detect as little as 2 methylated CpG dinucleotides per fragment. |
| What is the reproducibility of NimbleGen DNA methylation analysis arrays? |
While not fully validated by NimbleGen scientists, Weber et al. (Nat. Genet. 2007, 39(4):457-466) observed highly reproducible DNA methylation data between biological replicates (R = 0.92 for WI38 cells and R = 0.95 and 0.91 for sperm) when using MeDIP coupled with NimbleGen human promoter arrays. |
|
|
|
|
|
