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Roche NimbleGen provides the most sensitive ChIP-chip (chromatin immunoprecipitation on chip) microarrays and services on the market. These arrays enable you to rapidly identify the precise binding sites of specific DNA-binding proteins—such as transcription factors, histones, and polymerases—within a target genome, as well as allow you to look at chromatin remodeling in any region of a genome. NimbleGen ChIP-chip microarrays and services have become increasingly recognized as the platform of choice, with an expanding list of peer-reviewed publications.
Advantages 
Comprehensive Set of Array Designs Cover All Your Needs
Roche NimbleGen offers whole genome, promoter, and custom array designs, allowing you to choose the design that meets your research requirements. Our whole-genome ChIP-chip designs interrogate the non-repetitive regions of human, mouse, Arabidopsis, rat, dog, chicken, worm, fly, yeast, and E. coli genomes at intervals of 100bp or less for unbiased discovery of promoter/enhancer elements, transcription factor binding, histone modification/replacement, and DNase-I hypersensitivity. NimbleGen human and mouse promoter array designs, based on the latest public genome builds, tile across the promoters of known gene transcripts. You can also order an array design utilized by the ENCODE consortium or tailor an array specific to your experimental design.
High-Resolution Tiling = High-Resolution Detection of Binding Sites
NimbleGen high-density microarrays are available in three formats: 2.1M (2.1 million probes on a single glass slide), 385K (385,000 probes on a single glass slide) and 4x72K (test up to 4 samples per 72,000 probe array on a single glass slide). This high density provides for high-resolution tiling of your research genome and precise mapping of protein binding sites. With 2.1M arrays, the entire non-repetitive human and mouse genomes can be surveyed at 100bp intervals, each with a set of 10 arrays. Now with 4x72K arrays, you can increase sample throughput and lower cost per sample for targeted ChIP-chip analysis.
High Sensitivity and Specificity Provide Unparalleled Results
Roche NimbleGen's proprietary, light-mediated synthesis process produces high-density
microarrays of long oligonucleotide probes (50-75mer). These long oligo arrays, when used in combination with high-stringency hybridization protocols, produce results of unparalleled sensitivity and specificity. In addition, because Roche NimbleGen performs ChIP-chip experiments in a two-color protocol, where control and test samples are co-hybridized to the same array, inter-array variation is eliminated. As a result, NimbleGen ChIP-chip service can readily detect enrichment as low as two-fold of the target binding site in a ChIP sample, which can be extremely challenging for other array platforms to match.
Figure A. Identification of RNA Polymerase II Binding Sites in MCF7 cells using a HG18 RefSeq Promoter array. NimbleGen's graphical output enables you to visualize protein/DNA interactions.
Easy-to-use Graphical View of Data Aids Discovery
Roche NimbleGen SignalMap software enables you to visually interpret your data and perform peak finding. SignalMap’s graphic representation of your data aids the discovery of promoter/enhancer elements, transcription factor binding, and histone modification/replacement, and DNase-I hypersensitivity. A free, 30-day demo version of SignalMap software is available for download.
Most Up-to-date Genome Builds Ensure Most Accurate Results
NimbleGen ChIP-chip designs are based on the latest genome assemblies and sequence
annotations to ensure comprehensive and accurate representation of the genome. In
addition, you can continue to access array designs based on past genome data builds,
which can be particularly useful for comparisons to prior studies.
Applications and References
| Applications |
References |
| ChIP-chip Performance |
| To learn more about the performance of the NimbleGen ChIP-chip platform, review the first ever objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. |
Johnson DS, et al. “Systematic evaluation of variability in ChIP-chip experiments using predefined DNA targets,” Genome Res.: 18:393-403 (2008) |
| Transcription Factor Binding |
| Analyze transcription factor binding from human to A. thaliana using targeted promoter arrays or map factor binding across the entire genome of your choice in an unbiased manner. The combination of isothermal long oligonucelotide probes and high-density make detecting ultra-low fold-changes (less than 2-fold) a reality. |
Hatzis P, et al. “Genome-wide pattern of TCF7L2/TCF4 chromatin occupancy in colorectal cancer cells,” Mol. Cell Biol.: 28(8):2732-44 (2008) |
| Krishnakumar R, et al. “Reciprocal binding of PARP-1 and histone H1 at promoters specifies transcriptional outcomes,” Science: 319(5864), 819-821 (2008) |
| Parelho V, et al. “Cohesins functionally associate with CTCF on mammalian chromosome arms,” Cell: 132(3), 422-433 (2008) |
| Johnson DS, et al. “Systematic evaluation of variability in ChIP-chip experiments using predefined DNA targets, Genome Research: 18(3), 393-403, (2008) |
| Xu X, et al. “A comprehensive ChIP-chip analysis of E2F1, E2F4, and E2F6 in normal and tumor cells reveals interchangeable roles of E2F family members,” Genome Research: 17(11), 1550-1561, (2007) |
| Chromatin Structure |
| Interrogate chromatin structure by using ChIP-chip to analyze histone modifications, histone replacement patterns, and the precise positioning of individual nucleosomes at unprecedented resolution. Due to the inherent flexibility of NimbleGen ChIP-chip arrays, resolution can be defined as low as 1bp to reliably detect even the smallest perturbations within chromatin structure. |
Kirmizis A, et al. “Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation,” Nature: 449(7164), 928-932 (2007) |
| Rinn JL, et al., “Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs,' Cell: 129(7), 1311-1323 (2007) |
| Heintzman ND, et al. “Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome,”Nature Genetics: 39(3), 311-318 (2007) |
| Ozsolak F, et al. “High-throughput mapping of the chromatin structure of human promoters,” Nature Biotechnology: 25(2), 244-248 (2007) |
| Discovery of Genomic Elements |
| Use ChIP-chip as a tool for identifying and characterizing novel genomic elements and active promoters and enhancers by performing ChIP against RNA polymerase, transcriptional pre-initiation components, and histone modifications. |
Parelho V, et al. “Cohesins functionally associate with CTCF on mammalian chromosome arms,” Cell: 132(3):422-33 (2008) |
| Kim TH, et al. “Analysis of the vertebrate protein CTCF-binding sites in the human genome,” Cell: 128, 1231-1245 (2007) |
| Barrera LO, et al. “Genome-wide mapping and analysis of active promoters in mouse embryonic stem cells and adult organs,” Genome Research: 18(1), 46-59 (2007) |
| Kim TH, et al. “A high-resolution map of active promoters in the human genome,” Nature: 436(7052), 876-880 (2005) |
| DNAse Hypersensitivity |
| Detect and characterize regions of genomic DNA that are highly accessible to cleavage by DNase-I to identify open chromatin regions that permit gene expression. |
Boyle PA, et al. “High-resolution mapping and characterization of open chromatin across the genome,” Cell: 132, 311-322 (2008) |
| Follows GA, et al. “Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites,” Genome Research: 16, 1310-1319 (2006) |
| Crawford GE, et al., “DNase-chip: a high resolution method to identify DNase-I hypersensitive sites using tiled microarrays,” Nature Methods: 3(7), 503-509 (2006) |
| Sabo PJ, et al. “Genome-scale mapping of DNase I sensitivityin vivousing tiling DNA microarrays,” Nature Methods: 3(7), 511-518 (2006) |
Array Designs
Roche NimbleGen has a wide range of ChIP-chip 2.1M, 385K and 4x72K designs to choose from, or you can customize the array probe set to your specifications. The existing designs include whole genome survey sets, consisting of uniform tiling arrays covering all unique regions of the human genome, and promoter array designs aimed at known promoter regions. For custom designs, researchers can specify their regions of interest for a fine-tiling approach or design their own targeted probes for a tailored array solution.
Whole-Genome Designs
- 2.1M Whole-Genome Sets offer a higher density format so that fewer arrays are needed to analyze the whole genome of complex organisms at high resolution. Whole-genome formats provide two alternatives: a 10-array set at 100bp probe interval or a 4-array set at >200bp probe interval. For model organisms, a single 2.1M array slide has enough capacity to tile through the entire genome (55bp probe interval for D. melanogaster and 40bp probe interval for C. elegans).
- 385K Whole-Genome Sets tile uniformly across all unique regions of a genome at an average probe spacing of 100bp or less. Human, mouse, Arabidopsis, rat, dog, chicken and other genomes are currently available. Interrogate the whole genome or ENCODE regions to discover protein/DNA interactions in an unbiased manner.
Promoter Designs
Roche NimbleGen offers five types of promoter designs to suit your ChIP-chip analyses:
- 2.1M Deluxe Promoter Sets are single array designs containing expanded promoter regions for all known and alternative transcript start sites in human and mouse. In addition, these arrays tile through all annotated CpG islands, all annotated miRNA promoters, and manually selected ENCODE regions (human only).
- 385K Two-Array 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 Single Arrays
- 385K RefSeq Promoters are single array designs containing all known well-characterized RefSeq genes. The promoter regions on this array are covered by 50-75mer probes with approximately 100bp spacing, dependent on the sequence composition of the region. Designs are available for human and mouse.
- 385K RefSeq XM Promoters 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 Promoters are currently available for Arabidopsis thaliana.
Targeted Designs NEW!
Our recently released 4x72K ENCODE design offers a higher throughput, lower cost solution for testing the quality of ChIP-chip samples before moving to large-scale studies, or for screening focused targeted regions of interest, for up to 4 independent samples per slide. In addition we offer two 385K Human designs that target specific regions of interest.
Custom Tiling Arrays
For a completely tailored ChIP-chip array, customer specified genomic regions of interest are tiled with the same stringent probe selection methodology as used in NimbleGen’s catalog designs at the desired tiling density. Please contact Roche NimbleGen Sales for a quotation or contact Roche NimbleGen Technical Support with any questions about ChIP-chip custom tiling arrays.
Array Formats
| |
2.1M |
385K |
NEW!
4x72K* |
| Arrays per Slide |
1 |
1 |
4 |
| Total Number of Probes |
2,100,000 |
385,000 |
4 x
72,000 |
| Feature Size |
13μm x 13μm |
16μm x 16μm |
16μm x 16μm |
| Array Size |
62mm x
14mm |
17.4mm x
13mm |
7.8mm x
5.7mm |
| Slide Size |
1 x 3 in. (25 x 76 mm) |
| * Available for delivery only. |
Availability
2.1M, 385K, and 4x72K array formats are not available for every ChIP-chip array design. Please consult the Availability Guide on this page for a complete list.
Customer Workflows
ChIP-chip Delivery Workflow
Customers purchase catalog arrays or custom arrays from Roche NimbleGen, and perform the array experiments at their own laboratories or core facilities. NimbleGen arrays are synthesized on standard-sized glass microscope slides and are compatible with a range of hybridization, washing and scanning instrumentation. Roche NimbleGen provides a complete user’s guide to support customers with sample processing, array hybridization, scanning, data extraction, and analysis. Contact Roche NimbleGen for a list of required equipment and reagents, including Sample Tracking Controls for confirming sample identity on a 4x72K array. Roche NimbleGen NimbleScan and SignalMap software enable the same data analysis and visualization to be performed as in the Array Service mode. A free, 30-day demo version of SignalMap software is available for download. Roche NimbleGen also offers a training program to get you up and running with NimbleGen arrays quickly.
ChIP-chip Service Workflow
Another option to access ChIP-chip analysis from Roche NimbleGen is full microarray service. ChIP-chip service consists of the following steps:
- The customer selects a catalog ChIP-chip design or works with the Roche NimbleGen Bioinformatics team to create a custom array design. Roche NimbleGen manufactures the array.
- The customer prepares his or her samples, including immunoprecipitation and amplification, according to recommended protocols and ships these samples to Roche NimbleGen. For recommended sample prep protocols, see www.chiponchip.org or the UC Davis Genome Center.
- Roche NimbleGen labels the samples, performs the hybridization, scans the array, extracts the data, and performs a preliminary data analysis.
- The customer receives the raw data, scaled log2-ratio data, peak data, promoter reports mapping peaks to genes, genome annotation, and complete NimbleGen array design documentation.
Sample Requirements for 2.1M Arrays
| Sample Required |
6.5μg each IP and control samples |
| Sample Concentration |
250-500ng/μl |
Sample Requirements for 385K Arrays
| Sample Required |
4μg each IP and control samples |
| Sample Concentration |
250-500ng/μl |
Availability
Delivery and Service Workflows are not available for every ChIP-chip array design. Please consult the Availability Guide on this page for a complete list.
Availability Guide
Software
Roche NimbleGen NimbleScan software for data extraction provides tools specifically developed for detecting regions factor binding/histone modification from ChIP-chip array data. For each array, these tools generate a list of regions enriched by that antibody (peaks) and produce reports that map peaks to specific gene promoters. For more detailed information see the ChIP-chip Microarrays and Services datasheet.
NimbleGen SignalMap software enables you to visually interpret the scaled log2-ratio and peak data generated by NimbleScan. Graphical representation of your data aids the discovery of promoter/enhancer elements, transcription factor binding, and histone modification/replacement, and DNase-I hypersensitivity. A free, 30-day demo version of SignalMap software is available for download.
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 |
| Experimental Design |
| Do you have a recommended protocol for front-end sample processing for producing ChIP DNA? |
Yes, the protocol that we currently provide is based off a protocol from the Ren laboratory at UCSD. Customers are free to use whichever protocol they would like, but we highly recommend this protocol due to its reproducibility with ChIP-chip arrays. Contact Roche NimbleGen Technical Support if you would like the link to this protocol. |
| Does Roche NimbleGen perform front-end sample processing (e.g. IP and amplification)? |
No, Roche NimbleGen is not currently set up to perform contract ChIP-chip experiments for our customers. |
| What is the minimum number of cells required to perform a Roche NimbleGen recommended ChIP-chip experiment? |
The minimum number of cells used for a successful ChIP-chip experiment is approximately 1 x 107. However, ChIP reactions in which abundant molecules are being immunoprecipitated (e.g. histones and RNA polymerase II) require a lesser number of cells for a successful experiment. Remember, if you are starting with less material you would need to adjust all of the volumes and concentrations in each protocol. |
| What is a suitable negative control for ChIP-chip experiments? |
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 ChIP-chip. 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 other types of controls are used in a ChIP-chip experiment? |
The most common experimental control used when performing ChIP is an isotope control, such as nonspecific IgG or antibodies against GST or GFP. A potential pitfall in using these controls is that since the antibodies do not immunoprecipitate the nonspecific DNA yield is often very low. The resulting hybridization also tends to be much noisier and can result in many false positives due to amplification of trace amount of nonspecific DNA. Another, yet rare, control that is sometimes performed is a ChIP using uncrosslinked chromatin. However, many researchers prefer to perform ChIP with an antibody against the protein of interest in a cell line where the protein has been depleted (by target genetic deletion or siRNA). Alternatively, a cell line that does not express the protein of interest could be used as a negative control. |
| Is it possible to use more than one antibody when performing ChIP or do you recommend using only one antibody per procedure? |
Yes, many researchers combine multiple antibodies in their ChIP reaction to screen for antibodies that work well. If positive results are observed from this combined antibody approach, one can go back and perform ChIP using individual antibodies against the protein of interest. You can also perform experiments to examine the binding sites for a multi-protein complex by using a pooled sample of antibodies against all subunits of the complex. |
| What types of beads should I use to capture the immunoprecipitated DNA? |
There are two competing platforms for ChIP based on what type of beads one uses for immunoprecipitation: agarose protein-A/protein-G beads or magnetic latex immunoglobulin beads. Due to the inherent porosity of the agarose beads, a significant amount of nonspecific DNA binding is observed and consequently a much higher ChIP DNA yield than with magnetic latex beads. Some researchers have claimed that they observe good ChIP results without amplification using agarose beads. However, we recommend magnetic beads because we believe these beads will give much cleaner results. |
| What should I use as a reference sample? |
The majority of our customers use total (input) sample as a reference. Using a nonspecific IgG sample is not a suitable reference. |
|
| Array Design |
| Are the probes designed from both strands? |
No, Roche NimbleGen only designs probes based off of the forward strand. |
| How does Roche NimbleGen address repetitive elements in the genome for ChIP-chip designs? |
When available, we utilize conventional repeat masking, as done by the RepeatMasker program http://www.repeatmasker.org/). However, NimbleGen has no access to the repeat libraries necessary to perform this application, so we rely on third parties to supply this type of masked sequence. However, we find that RepeatMasker is often overly aggressive and can mask 50%-55% of human DNA sequence. We have developed our own method of repeat masking which is dependent on the mean frequency of the 15mers which make up each 50mer oligo. A table is made of the count of all 15mers that appear in the genome, from both strands. Then a 15mer window is slid along each oligo, looking up the count of each 15mer 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. A similar technique is used by other groups. 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. |
| Why do you use 100bp spacing in ChIP-chip designs? |
Our research/development staff has performed experiments in which human probe spacing is varied; they observe a much poorer signal/noise ratio as well as a dramatic increase in the number of false positives when spacing is greater than 100-120 bp. |
| What spacing do you recommend for ChIP-chip designs? |
We recommend probe spacing of 100bp or less. |
| What are the human ENCODE biologically significant regions that are tiled on 4x72K and human 2.1M Deluxe Promoter arrays? |
These regions were picked manually by the ENCODE consortium and are deemed biologically significant in terms of transcriptional regulation. The regions include the HOXA cluster (chromosome 7), ß-globin cluster (chromosome 11), and others. |
|
| Sample Processing |
| How much IP sample should be expected prior to the amplification step? |
The amount of IP sample obtained truly depends on the antibody quality and amount of starting material. A standard ChIP reaction yields DNA fragments in the range of ~100ng. |
| Do I need to amplify my ChIP samples? |
Whole genome amplification (WGA) or ligation mediated-PCR (LM-PCR) needs to performed when there is less than 4µg ChIP DNA. |
| What method should I use to amplify my ChIP samples? |
Past and present NimbleGen ChIP-chip customers have had very good experiences working with the Sigma WGA kit (#WGA2-50RXN). The WGA method seems to be easier and the quality of the amplified DNA is quite good. Many customers find that it is difficult to get LM-PCR to work well. For those that are just beginning ChIP-chip studies, we recommend WGA. |
| What yield should I expect from LM-PCR? |
A standard ChIP reaction yields less than 100ng DNA. After one round of LM-PCR amplification that yield can increase to a few micrograms. If more DNA is needed, a second round of amplification can be performed. |
| Does LM-PCR cause bias in the sample? |
We have found that there tends to be more bias with samples amplified by LM-PCR when compared to WGA. We recommend running the amplified DNA on an agarose gel to check for the presence of multiple DNA bands, which indicates that the sample is of poor quality. Generally, DNA amplified by WGA gives a smear rather than multiple bands. |
| Can I use T7 amplification for generating ChIP DNA? |
Yes, but we have found that customers need to adequately remove all RNA and protein from the sample. T7 amplified samples frequently arrive at Roche NimbleGen with RNA and protein contamination, which results in low labeling yields and subsequently less than adequate amounts of labeled sample to perform an array hybridization. |
| Why does NimbleGen use 7mer primers for short fragment labeling? |
We use 7mer primers because they seem to be much more efficient at labeling short DNA fragments (<200 bp). We consistently have trouble obtaining adequate yields for short DNA fragments when using 9mer primers. |
| What is the difference between 7mer and 9mer labeling for the same samples if it is above 200bp? |
We are still unclear on the differences between 7mer and 9mer sample labeling for larger DNA fragments. We are in the process of running experiments to test for any differences. Preliminary data has shown that the differences are quite small. |
| Will all ChIP DNA eventually be labeled using 7mer primers? |
Labeling ChIP DNA using exclusively 7mer random primers is a possibility. We are in the process of validating 7mer labeling for ChIP-chip. As of now, all ChIP samples should still be labeled using 9mers. |
| What is the expected yield from labeling reactions? |
One microgram each of IP and total sample are labeled with Cy5 or Cy3, respectively, using a 9mer primer. Our labeling procedure yields 28µg +/- 10µg for ChIP-chip samples. Samples <200bp yield an average of 10µg per labeling reaction. |
| Which dye should I use for my experimental and reference sample? |
We normally label the total sample (input) with Cy3 and the IP sample with Cy5. In the case that you would want to perform a dye swap experiment, you would label total sample (input) with Cy5 and the IP sample with Cy3. |
| Do I need to order another chip if I am going to perform dye swaps or co-hybridize DNA immunoprecipitated using a nonspecific antibody control? |
Yes, to perform a dye swap or co-hybridize DNA immunoprecipitated using a nonspecific antibody control you would need to order additional chips. |
|
| Sample Requirements |
| What are the sample requirements for ChIP-chip? |
We require 4µg DNA 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. |
| What is the normal ChIP-chip fragment size? What if my fragments are smaller? |
DNA shearing from a typical ChIP experiment yields 200-1000bp fragments. If the majority of DNA fragments are below 200bp, Roche NimbleGen will have difficulty obtaining adequate labeling yields and hence may not have sufficient material to hybridize to the arrays. However, if your fragments are between 100-1000bp, with the majority of fragments >200bp, than your samples should work well. |
| How much ChIP DNA do I need to supply if I order the 10 array set? |
The 10 array set requires approximately 65µg DNA. For a more economical choice for analyzing whole genome protein/DNA interactions, you can use the 4 array set (human and mouse), which requires at least 26µg DNA. |
| What if my sample concentration or yield is less then required? |
If your sample does not meet our QC requirements you will be contacted by Roche NimbleGen for replacement samples. If you are unable to supply replacements you still have the option of proceeding with the experiment; however, there may be extra charges attached to this sample and the success of your experiment will not be guaranteed. |
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| Data Analysis |
| Does Roche NimbleGen normalize ChIP-chip data? |
No, there is no normalization of ChIP-chip data. However, we do scale the GFF files by subtracting the bi-weight mean for the log-ratio values from each log-ratio value. |
| Does Roche NimbleGen scale ChIP-chip data? |
Yes, Roche NimbleGen scales the ratios in the .gff files by subtracting the bi-weight mean for the log-ratio values from each log-ratio value. If you would like more information about how to calculate a Tukey bi-weight mean scaling please go to Bi-Weight Scale. |
| Why does Roche NimbleGen use Tukey's bi-weight function for scaling ChIP-chip data? |
RMA looks at all the arrays in a set and normalizes the data for all the arrays. ChIP-chip is a two color array with the reference on the array and is therefore thought of as a stand alone experiment. RMA is not an appropriate analysis for ChIP chip data. The Tukey bi-weight function is used to account for differences in the dyes on the array, whereas RMA is used to account for differences between arrays so that the arrays can be compared. |
| Are there any statistical tests that are applied to my ChIP-chip data? |
Yes, we determine the false discovery rate (FDR) for each peak identified from the scaled log2-ratio data. First the scaled log2-ratio data is analyzed to identify peaks above a specified cutoff value. Assume we find 20 peaks that are above this cutoff value; the scaled log2-ratio data is then randomized 20 different times and after each permutation it is determined how many peaks are still above the cutoff value. So, if after randomizing the data 20 times we find that there are 2 peaks above the cutoff value, the FDR is 10% (which is a reasonably good FDR value). FDR values can differ depending on the peak height and number of probes comprising the peak. On SignalMap, the peaks will be color coded corresponding to FDR value for quick identification of statistically significant peaks. |
| Other than the pair files, does Roche NimbleGen supply any other raw data files? |
No, pair files are the only raw data files that Roche NimbleGen includes in delivered ChIP-chip data. |
| What are the background measurements for a ChIP-chip array? |
For any ChIP-chip array, the signal is a mix of non-specific signal, “background”, and specific signal. The information given from random probes do not represent true background measurements, but rather non-specific binding events. Currently, we do not calculate background. |
| Is there another way to analyze ChIP-chip data? |
Yes, the following five sites have been developed to analyze ChIP-chip data.
1. M-peak: Nature. 2005 436(7052):876-80
2.TAMALPAIS Server: 2006 Genome Research 16:595.
3.ACME (in R language): Methods Enzymol. 2006;411:270-82.
4.ChIPOTle: Genome Biology 2005, 6:R97. For the Perl version, go to ChIPOTle Peak Finder
5.Model-based Analysis of 2-Color Arrays MA2C. |
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| Deliverables |
| Can I get the images for my data? |
Yes, we can supply 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 catalog design so I can see what regions of the genome have coverage? |
Yes, we can generate this information in GFF 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. |
| Does Roche NimbleGen generate reports listing the most significant binding/modification events for my ChIP-chip experiment? |
Yes, we are including two promoter reports that map the peaks from your ChIP-chip 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 lists the approximate location of the peak as a negative position (upstream of the start site) or positive position (downstream). Also listed are accession number of the gene, gene ID, chromosome position, among others. These reports narrow the genomic regions to look at when moving forward to validate your ChIP-chip data (e.g. gel mobility shift assay). |
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| Capabilities |
| What if a customer has non-nucleosome samples that have an average length of <200bp? |
If non-nucleosome samples are <200bp, we utilize 7mer random primers to label DNA. However, at this time such samples will be marked as “at risk” because there has not been sufficient validation using 7mers for non-nucleosome samples. |
| Can I use NimbleGen ChIP-chip to map DNase I hypersensitive sites? |
Yes, NimbleGen ChIP-chip arrays can be used to map DNase I hypersensitive sites. A manuscript by Crawford et al. (2006. Nature Methods. 3:503-509) describes the use of NimbleGen tiled arrays to map hypersensitive sites. Other manuscripts mapping hypersensitive sites using NimbleGen arrays include Sabo et al. (2006. Nature Methods. 3:511-518) and Follows et al. (2006. Genome Research. 16:1310-1319). |
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