NimbleGen Systems, Inc. - Frequently Asked Questions

All Topics

Array Delivery

Can array delivery customers reuse the arrays?

Array delivery customers can strip CGH, ChIP-chip, and CGS arrays. We have not optimized Expression array re-use yet.

Array Design

Are our oligos capped for termination in case nucleotides are not incorporated, or are they uncapped permitting internal "deletions"?	After synthesis we do a deprotection step to uncap the last base.
Can I order an array design that was made for another customer?	Yes, if the customer who originally ordered the array provides written consent.
Do genomic positions differ from build to build?	Different builds, different positions. There will likely be some overlap, but probably not 100%. Can use LiftOver tool @ UCSC to convert positions from one build to another.
How does NimbleGen calculate Tm of probes?	If an isothermal design is requested, we screen probes with one of two algorithms, depending on probe length, for a target Tm of 76C. For probes greater than or equal to 36 nucleotides we calculate the melting temperature of substr(seq, start, length) using the formula from Bolton and McCarthy, PNAS 84:1390 (1962) as presented in Sambrook, Fritsch and Maniatis, Molecular Cloning, p 11.46 (1989, CSHL Press). Tm = 81.5 + 16.6(log10([Na+])) + .41*(%GC) - 600/length. Where [Na+] is the molar sodium concentration, (%GC) is the percent of Gs and Cs in the sequence, and length is the length of the sequence. For shorter oligos, we use the melting temperature of the given oligo, as calculated using eqn(ii) in Rychlik, Spencer, Roads, Nucleic Acids Research, vol 18, no 21, page 6410, with tables of nearest-neighbor thermodynamics for DNA bases as provided in Breslauer, Frank, Bloecker, and Markey, Proc. Natl. Acad. Sci. USA, vol 83, page 3748.
How many copies of the same probe are in the same location on an array?	We estimate that there are about 1x10⁶ probes of the same sequence on each feature of our array.
How many probes are included on an array?	1 plex = 385K 4-plex = 4 x 72K
Can I order a design from an older catalog even if it does not appear in the current (2006) catalog?	2006 catalog arrays were done using new specifications. Some of the old designs did not meet those specifications and therefore can no longer be ordered as catalog designs.
What should I include as additional QC probes on my custom design?	Some people put positive and negative controls on the array. Positive controls, for example where you have a specific sequence known to be present in the sample (or spiked in). Negative controls where you include probes that should not hybridize.

Data Analysis

Can we transform the log ratio in GFF format to estimate fold differences?	Yes.
What does a failure in data analysis mean?	It means that during the data analysis the array did not meet our QC scores.

Miscellaneous

Can I obtain strand specific information?	Our labeling protocol is an amplification protocol that uses Klenow and produces sense and anti-sense labeled probes. It does not allow us to obtain strand specific information for service customers. Array delivery customers can obtain strand specific information by ordering arrays and end-labeling their samples.
Can NimbleGen arrays be scanned using the Agilent scanner?	The 5 micron Agilent scanner can work with NimbleGen arrays.
Do I need new bar codes for my sample replacements?	Not for replacement samples for an existing order. Replacements / repeats that are being done in a new OID should have new bar codes.
Does NimbleGen supply design files for the arrays that I order?	Yes.
How does NimbleGen synthesize arrays?	NimbleGen uses a proprietary in situ DNA synthesis method based on light modulated oligonucleotide synthesis. To control where light strikes the array, NimbleGen has adapted a Digital Micromirror Device (DMD, Texas Instruments, Inc.). The DMD is an array of tiny, individually addressable polished mirrors that can be positioned to either reflect the UV light source on a known position within the array or redirect light away from that position. By coordinating the sequence of illumination and addition of photoprotected phosphoramidites, the technology has the capability of synthesizing more than 385,000 user-defined oligonucleotide probes with high yield and high accuracy.
How is NimbleGen array synthesis different from spotted array technology?	Spotted array technologies are based on the physical deposition of biomolecules (typically oligonucleotides or cDNAs) within the array. The deficiencies of spotted array technologies include significantly lower feature density, the complexity of synthesizing, purifying and cataloging and tracking all biomolecules used for spotted array manufacturing, and the difficulty of making iterative design changes. Any errors in the dispensing of biomolecules into source plates will carry through to the final manufactured array. Additional errors can be caused by incorrect annotation or labeling of source tubes or plates, plates used out of sequence during the spotting process, etc. By contrast, NimbleGen microarrays are synthesized directly on the slide surface using only the source sequence information and the synthesis chemistry. This approach eliminates all of the laborious and potentially error prone steps required for the preparation of spotted arrays. In addition, the small feature size possible with NimbleGen microarrays means that many more features (more than 350,000) are available on a single array.
How is NimbleGen's technology different from other in situ technologies?	Other commercially available in situ synthesis technologies suffer from either low feature density or poor design flexibility making them less attractive for custom DNA array synthesis. The predominant method is based on the photochemical synthesis of high quality arrays, however the method relies on the use of chromium lithographic masks, rather than an addressable mirror array, to generate the desired light patterns for array synthesis. Since the preparation of physical masks is an expensive process, the correction of error in array designs and the preparation of custom arrays are costly. Other methods rely on the sequential deposition of phosphoramidites or deprotection reagents within the growing array to control synthesis in a site specific manner. While these methods have some of the flexibility advantages of NimbleGen arrays, they are ultimately limited by the print resolution of their deposition process. As a result the total number of probes available is typically 10 fold lower.
Is our nLIMS system for sale to our customers?	No.
What is the average dynamic range that we get with our system?	We do not have a dynamic range that we have researched. We can detect a 2-fold difference and have seen a log₂ ratio as high as 8. Our observed Log₂ dynamic range is 2 8.
What is the turn around time for a complete NimbleGen array assay?	Once an array design is complete, NimbleGen can manufacture and QC a microarray in less than two days. A complete hybridization experiment (sample prep, labeling, QC, hybridization, and analysis) can be accomplished in less than a week. Actual turn around time for your samples will be determined by our production queue.
When do you manufacture my array?	Each array is manufactured when the relevant sample is received in our Iceland facility.

Sample Processing/Requirements

How can I concentrate my sample?	SpedVac or PCR purification column, eluting in less volume.
Do I need to use screw cap 1.5ml tubes to submit my sample?	NimbleGen recommends National Scientific Supply BioStor™ Screw-Cap Vials (1.6ml) (cat # BC16NA-PS). Can use flip top tubes.
How do I know if my organism is Class I, II, or III?	Currently need to contact Tech Support.

Service Process

Can NimbleGen pool my samples?	Yes.
If one of my samples is held up due to sample QC, labeling, etc, is the rest of my order also held up?	No.
What is the composition of your buffers?	NimbleGen will not provide the composition of our hybe buffer or wash buffers in our hybridization kits. The composition of our buffers is proprietary.
Why is the hybridization temperature 42°?	NimbleGen has optimized our hybridization temperature and our hybridization buffers to best hyb sample on our arrays with the probe lengths we use. We do not have a technical note explaining our hyb temp setting but we have done the experiments in our lab to find the best temperature for our arrays.

Software

How do I search for a specific gene when using SignalMap?	Use the search function under Edit in SignalMap and put in the name of your gene. You will need to use the NM number or a number unique to the transcript because many gene names have several transcript sites. Also, you will need the annotation file loaded which should have come with your data. The annotation file is in the GFF folder and usually will have a date followed by some other notation. It is also the only file that does not have a chip number. Our SignalMap users guide which comes on the SignalMap CD should also be able to help you figure out how to use the search function and how to find the annotation file.
Is NimbleGen software compatible with PC and MAC's?	Compatibility is as follows. Nimblescan: compatible with Microsoft® Windows® 2000 or later, Apple MacOS 10.3 or later, and Linux (2.4 kernel or later). SignalMap: compatible with Microsoft® Windows®.
What is window size (SignalMap)?	The window size is the number of bases that the program looks at when running the second derivative. There for if you window size is set at 500bp and you have 100bp spacing you should have about 5 probes that the peak finding will check to see if there is a peak in that window.
What other software programs can be used for data analysis?	Spotfire, GeneSpring, R, MATLAB.
What text editor programs do you recommend?	emacs, wordpad, metapad
Will the software associated with Agilent scanners give data files that can be used with NimbleScan?	It will probably work if the following conditions are met: 1. All images in the TIF must be exactly the same width and height, implying they come from multiple channels of the same scan. There can't be low res versions or thumbnails in the file. Just quantifiable scans. 2. All "channels" must be digitized as 16 bit grayscale where zero implies no signal and 65535 max signal. 3. To get auto-alignment to work the images must be spatially calibrated. IOW, the size of a pixel is specified in the TIF file. 4. We assume the signal response is linear. IOW we assume twice as bright means twice as much stuff. 5. The images must be spatially linear to within a couple microns or so. We have to be able to lay down a four point alignment and get accurate registration across the field of view.

Sequence Capture

Experimental Design

How much sequence can I capture on your current 385K custom arrays?	The maximum amount of sequence that our current NimbleGen Sequence Capture arrays can capture is 5Mb.
What organisms does Roche NimbleGen currently accept for NimbleGen Sequence Capture service?	At this time, we are only accepting genomic DNA from human and mouse. In principle this method should work with any species where a sequenced genome is available, and we continue to work on developing and evaluating optimized protocols for both services as well as products that will enable capture of targeted DNA from other species. If you are interested in developing your own protocol for use of NimbleGen products or services with other species, we strongly recommend performing initial pilot studies before embarking on large-scale projects.
What types of sequence are researchers typically capturing when applying this technology to their research?	The types of sequences that researchers are capturing typically fall into two distinct categories: discontiguous and contiguous. Examples of discontiguous regions include exons, promoters, and enhancers. A classic example of a contiguous region would be a disease associated region (DAR), such as the BRCA1 locus, in which you could look at different intervals sequence coverage around the gene.
Why should I use NimbleGen Sequence Capture microarrays instead of various PCR methods as a preparative tool for next-generation sequencing?	The severe costs, performance limitations, and extensive amount of labor required for large-scale PCR experiments makes taking full advantage of the capacity of next-generation sequencers virtually impossible. With NimbleGen Sequence Capture arrays, you can reduce the complexity of your genome in a matter of weeks all while saving considerable time and money.
Are there any publications demonstrating the reproducibility and robustness of NimbleGen Sequence Capture technology?	Yes, there are an ever-increasing number of publications. Click here to view the list of current publications citing the use of NimbleGen Sequence Capture technology.

Array Design

How do I go about designing a custom NimbleGen Sequence Capture array?	Once you place an order, you will complete our Design Specification form indicating what regions (chromosome, tiling start position, and tiling stop position) you would like tiled on the array. Once our Bioinformatics scientists have designed the array, they will send it to you for approval.
What genome builds of human and mouse are Roche NimbleGen using to design a 385K custom array?	We are using the latest genomic builds for human (HG18) and mouse (MM9).
Will I be able to design a custom NimbleGen Sequence Capture array that targets repetitive regions?	No, at this time we are only designing probes that cover unique regions of the human or mouse genome.
Who owns the designs for the sequence capture arrays?	The design that we create for each NimbleGen Sequence Capture array - whether that array is delivered to customers for their own use or we use it in performing a service for the customer - is proprietary to and the property of Roche NimbleGen.

Sample Requirements

What are the sample requirements for a NimbleGen Sequence Capture experiment?	We require at least 21μg human or mouse genomic DNA at a concentration of 250-500ng/μl. The A₂₆₀/A₂₈₀ ratio should be at least 1.8 And the A₂₆₀/A₂₃₀ ratio should be at least 1.9. Also, the genomic DNA should not show a smear when analyzed on a bioanalyzer.
What if my submitted genomic DNA samples are less then required?	If your samples do not meet our QC requirements you will be contacted by Roche NimbleGen for replacement samples.
Do you accept whole-genome amplified genomic DNA?	No, at this time we are only accepting unamplified genomic DNA.

Deliverables

After Roche NimbleGen captures my desired sequences, what do I get back?	You will receive 10mg of amplified DNA (by LM-PCR), which can be used directly for next-generation, high-throughput sequencing.
What types of QC information and supporting data files will I receive after my sequences are captured?	You will receive a report on sequence capture yield and the level of enrichment. Also included are a list of regions targeted by the array design, our SignalMap software, and a user's guide that describes how to sequence the captured DNA using the 454 GS FLX instrument.

Downstream Applications

Can I perform the Sequence Capture process in my own lab?	You can order our custom arrays and follow the published protocols in: Direct selection of human genomic loci by microarray hybridization (Nat Methods. 2007 Nov;4(11):903-5). Free PDF Download Please understand, however, that these protocols are not optimized, validated commercial products, and as such, Roche NimbleGen Technical Support staff may not be able to provide the level of technical support that accompanies a fully launched delivery product.
What regions can be captured by this technology?	These can be any regions in the genome, either contiguous, such as disease associated regions, or non-contiguous, such as exons of a candidate gene panel. Please note that, in our technology development efforts, we currently only design probes against unique parts of the genome, although some repetitive regions can be captured by the array and sequenced with long reads from 454 GS FLX technology if they flank unique regions. The total size of captured regions per array can vary from a few hundred kilobases to a few megabases using existing 385K feature arrays. With NimbleGen HD2 high density arrays (2.1 million features) and optimized protocols, a single array will be able to capture much more of the genome. For more information on the current technology status, please see: Direct selection of human genomic loci by microarray hybridization (Nat Methods. 2007 Nov;4(11):903-5). Free PDF Download
Will this technology be compatible with all next-generation sequencing platforms?	The Roche NimbleGen Sequence Capture method yields the highest quality results when used in conjunction with a sequencing technology that can deliver sequence read lengths in excess of 200bp because long reads enable comprehensive variant detection. To supply our customers with an affordable, high-quality solution, we have been working closely with our sister Roche business area, 454 Life Sciences, to develop, test, validate, and optimize protocols for obtaining enriched DNA that can be directly and easily integrated into the workflow of the 454 GS FLX instrument. The 454 GS FLX instrument delivers read lengths of 250bp and is the most appropriate sequencing technology for the NimbleGen Sequence Capture solution. Other early customers are working on modified protocols to enable use of NimbleGen Sequence Capture arrays and reagents with other sequencing platforms; however, these protocols have not been internally validated by Roche NimbleGen.
What is the cost for using this technology?	For most studies that require resequencing of large regions of the genome, this technology will clearly offer significant benefits in terms of cost and time, particularly when compared with multiplex and/or long-range PCR. Please contact your local Roche NimbleGen sales representative for a quote.

Future Developments

What will Roche NimbleGen offer in the future for NimbleGen Sequence Capture products?	In mid-2008, we will be offering NimbleGen Sequence Capture arrays for delivery. This allows you to perform capture experiments in your own lab or core with the use of a hybridization station, elution system, and a kit with all necessary reagents.
Will Roche NimbleGen offer NimbleGen Sequence Capture products on our HD2 (2.1 million features) platform?	Yes, we are planning to release NimbleGen HD2 arrays as part of the NimbleGen Sequence Capture product offering in mid-2008. With NimbleGen HD2 arrays, you can capture up to 30Mb of sequence, compared to 5Mb with the current 385K arrays.

CGH

General

How is NimbleGen array technology different from other platforms?	NimbleGen manufactures custom, high-density DNA microarrays using its proprietary Maskless Array Synthesizer (MAS) technology. At the heart of the system is a Digital Micromirror Device (DMD) that uses a solid-state array of miniature aluminum mirrors to create “virtual masks” that replace the physical chromium masks used in traditional arrays. The DMD directs a pattern of UV light projected onto a microscope slide, which when coupled with UV-mediated DNA synthesis in a parallel, combinatorial manner, can generate 385,000 to 2.1 million unique probes on a single microarray.
How is the version 2.0 aCGH a better design than version 1.0?	Segmental duplications are more reliably detected due to enhanced probe coverage in low-copy repeat regions on the version 2.0 product.
How many probes are included on a single array?	385,000 probes on our current array format; 2.1M probe format available in 2008.
Does NimbleGen offer aCGH in a multiplex format?	Our current 385K array format is available in 4-plex (72K).
What is the resolution of your arrays?	Effective resolution varies according to the spacing of individual probes (generally 5-10x the median probe spacing). Our human whole-genome designs provide 5-50 kb resolution depending on the array platform (385K vs. 2.1M feature format). Custom fine-tiling arrays enable greater probe density in specificied regions and breakpoint mapping down to 10-100bp.
Do NimbleGen array designs include non-coding regions of the genome?	Yes - NimbleGen offers unbiased tiling-path whole-genome designs that include genic and intergenic regions.
Can NimbleGen design an array tailored to my specific research needs?	Yes - specify regions of interest and NimbleGen will design a custom array to meet your research needs.
What resolution scanner do I need?	A scanner with 5μm resolution is required.
Can NimbleGen aCGH distinguish single vs. multi-copy amplifications?	Yes.

Sample Processing

What are the sample requirements for aCGH?	NimbleGen recommends 2.5μg of high-quality unamplified gDNA.
What protocol does NimbleGen recommend for DNA isolation?	Any protocol that provides high-quality gDNA.
What reference sample should I use?	There are several options for choosing a reference sample. For example, for cancer studies choose a patient's own DNA (e.g. tumor vs. germline), DNA from a single individual, or pooled DNA from several individuals (NimbleGen can provide pooled male or female DNA).

Array Design

Are NimbleGen designs up-to-date with the latest genome builds?	Yes, NimbleGen technology allows the rapid production of new array designs based on the latest genome builds.
Are designs based on earlier genome builds available?	Yes.
Does NimbleGen offer non-human aCGH designs?	Yes - NimbleGen offers catalog designs for a wide range of organisms. In addition, NimbleGen can generate custom designs for any genome (or region of genome) for which high-quality sequence is available.
Does NimbleGen use only non-unique probes?	Generally only unique probes are included in array designs. However, NimbleGen can easily generate array designs that contain probes representing non-unique regions of the genome (e.g. segmental duplication).

Data Analysis

What software do I need to view my data?	NimbleGen provides SignalMap data visualization software.
Can I analyze my data using other software programs?	Yes - NimbleGen data can be viewed and analyzed using a variety of commercially available software programs.
Can I view my data in the UCSC genome browser?	Yes.
How do I get gene annotation information?	NimbleGen provides a gene annotation track corresponding to your design. Additional annotation tracks can be viewed in the UCSC genome browser.

Array Delivery

Can I purchase NimbleGen arrays for use in my own lab or core facility?	Yes.
Which CGH designs are available for array delivery?	All catalog and custom designs currently available for array service.
What kits are availble for use with NimbleGen microarrays?	Hybridization, labeling, array re-use
Can I scan NimbleGen arrays using my Agilent scanner?	Yes.
What software do I need to scan and analyze my data?	NimbleGen provides array delivery customers with NimbleScan array scanning and analysis software.
Does NimbleGen offer training for array delivery customers?	Yes - NimbleGen offers training on-site or at our Madison, WI location.
Can I strip the labeled sample and re-hybridize the microarray?	Yes - NimbleGen offers an array re-use kit for stripping and re-hybridizing arrays.

ChIP-chip

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 protcol 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. Please contact NimbleGen Technical Support if you would like the link to this protocol.
Does NimbleGen perform front-end sample processing (e.g. IP and amplification)?	No, 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 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. 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, NimbleGen only designs probes based off of the forward strand.
How does 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 reseach/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.

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 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, 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 38 array set?	The 38 array set requires approximately 50µg DNA. However, if the majority of your sample consists of fragments <300bp, we advise that you to supply more than 50µg of sample.
What if my sample concentration or yield is less then required?	If your sample does not meet our QC requierments you will be contacted by 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.

Data Analysis

Does 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 NimbleGen scale ChIP-chip data?	Yes, 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 NimbleGen use Tukey's biweight 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 biweight 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 chips 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 NimbleGen supply any other raw data files?	No, pair files are the only raw data files that 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.

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 along with the visualization software SignalMap.
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 catalog designs. You will need a copy of SignalMap to view the GFF files.
Does 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 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 ChIP-chip data (e.g. gel mobility shift assay).

Capabilities

Do we support data analysis for nucleosome mapping?	Yes, we currently support data analysis for nucleosome mapping. We plan to release a nucleosome-chip product next year, which will include a fully optimized design and data analysis.
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).

DNA Methylation

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 have a CpG island plus promoter array that includes 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 SignalMap to view the GFF files.

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. NimbleScan 2.3 users can 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 along with the visualization software SignalMap.
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 catalog designs. You will need a copy of SignalMap to view the GFF files.
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.

Gene Expression

Multiplex

What is the advantage of the NimbleGen 4-plex array platform?	The NimbleGen 4-plex array platform allows simultaneous hybridization of 4 samples on a single slide. You can conveniently run either replicates of the same sample or multiple samples on the same slide, thus reducing cost.
How many features are on each array of the NimbleGen 4-plex format?	Each array on the NimbleGen 4-plex slide has 72,000 features.
Do you offer multiple probes per transcript in the NimbleGen 4-plex format?	Yes, each transcript is interrogated by 3 or more probes, depending on the complexity of the organism. Please see our catalog for more details for each organism.
What kind of controls do you include on each array to ensure accurate sample loading and to confirm the integrity of the experiment?	Each array contains probes for Sample Tracking Controls (STCs) that are used for the following purposes: a) To identify the samples after hybridization; each labeled cDNA sample is spiked with a unique Sample Tracking control before loading. b)To ensure that there is no sample mix up between arrays.
Do all arrays on a NimbleGen 4-plex slide have the same design?	Yes. Currently all arrays on a gene expression multiplex array have identical designs.

Array Design

Do you include microRNAs or non-coding RNA in your catalog designs for expression?	Currently, no microRNA or non-coding RNA regions are used for NimbleGen gene expression array designs.
What is the probe length in gene expression arrays?	NimbleGen expression arrays contain 60-mer probes.
Do you offer catalog and custom array designs?	Yes, we offer both catalog and custom array designs for differential expression analysis. Please check our website for the current catalog. If you do not see your organism, please contact the Account Manager for your region to discuss a custom design.
Are your gene expression probes designed with a 3' bias?	Yes, probes are designed with some 3' bias. Our expression arrays are designed by first determining the transcript terminator as annotated in NCBI. We then take 1500bp upstream of that location and design our probes within that region.
Which strand is represented on the array?	The sense strand is represented on our arrays.
Can NimbleGen design a custom expression tiling array for my specific research needs?	Yes, NimbleGen can design and deliver to you custom expression tiling microarrays with probe spacing and probe length that best fits your experimental goals. Please contact the Account Manager for your region to discuss a custom design.
Are NimbleGen designs up-to-date with the latest genome builds?	Yes, NimbleGen technology allows the rapid production of new array designs based on the latest genome builds.
Are designs based on earlier genome builds available?	Yes, please contact the Account Manager for your region.

Sample Preparation

What is the recommended protocol for isolating RNA?

We recommend the RNeasy Mini Kit (Qiagen, Cat. No. 74104) or the TRIzol Reagent (Invitrogen, Cat. No. 15596-026)). If using TRIzol-isolated RNA for cDNA synthesis, it is beneficial to perform a secondary cleanup step using RNeasy Mini Kit. Immediately after the ethanol precipitation step in the TRIzol procedure, proceed with the RNeasy Mini Kit according to the manufacturers recommendations.

Can I use a different RNA isolation method than recommended?

Any RNA isolation method that provides RNA which meets the purity and integrity criteria outlined by NimbleGen can be used.

How do you assess the quality of RNA and cDNA samples?

Pure and intact RNA and cDNA should have A260/A280 and A260/A230 ratios of at least 1.8. Additionally, they should appear intact when analyzed by gel electrophoresis or using a Bioanalyzer (Agilent).

How much RNA or cDNA do you need for gene expression analysis?

This depends on whether you are sending the samples to NimbleGen for full service or if you are doing the experiment in your lab, and whether your sample is from eukaryotes or prokaryotes. When performing experiments in your lab using NimbleGen-recommended protocol, 10 μg total RNA, 1 μg mRNA, or 1 μg cDNA is required. For service, the amounts required are listed in the table below.

	Total RNA*	mRNA*	cDNA
Eukaryote	20μg	10μg	4μg
Class I Prokaryote	20μg	-	4μg
Class II and III Prokaryotes	30μg	-	8μg
*Please note that the NimbleGen procedure for sample processing does not include RNA amplification.

What is the expected cDNA yield from 10μg total eukaryotic RNA?

Using our standard protocol, we routinely get at least 5μg cDNA from 10μg total RNA.

If I do not have 10μg of RNA, what method for RNA amplification can I use in my own lab?

We have successfully used the MessageAmp II aRNA Amplification kit (Ambion, Cat. No. 1751) for RNA amplification of two human reference samples: Universal Human Reference RNA (Stratagene, Cat. No. 740000) and Human Brain Reference RNA (Ambion, Cat. No. AM6050). As with any amplification, there is a risk of introducing bias into the experiment.

What kind of samples are recommended for hybridizing on NimbleGen gene expression arrays?

We recommend using Cy3-labeled cDNA sample on our gene expression arrays. Please contact the Account Manager for your region to obtain a recommended protocol for preparing Cy3-labeled cDNA.

Data Analysis

What method of normalization does NimbleGen use for gene expression?	We normalize expression data using quantile normalization. See Bolstad B, et al. (2003), A comparison of normalization methods for high density oligonucleotide array data based on bias and variance. Bioinformatics, 19:185-193.
How does NimbleGen generate gene calls from gene expression arrays?	Gene calls are generated using the Robust Multichip Average (RMA) algorithm. See Irizarry R. et al. (2003), Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:249-264.
What software do you recommend for further analysis of gene expression data?	NimbleGen provides gene expression values in calls file that can be conveniently imported into multiple gene expression analysis software such as ArrayStar (DNAStar). Currently, with the purchase of a full service project, you will obtain a 30-day demo version of ArrayStar along with your data. Array delivery customers can also request this 30-day demo version from NimbleGen.
Can NimbleGen gene expression data be used with publicly available software packages?	Several analysis packages that have been used with NimbleGen gene expression data are: 1. NMPP: Described in Bioinformatics (2006); 22: 2955 - 2957 2. MeV: Described in J. Exp. Biol. (2007); 210: 1507 - 1517. 3. R: Available at www.bioconductor.org, requires data in XYS format.

Array Service

How much RNA or cDNA do you need for gene expression analysis?

	Total RNA*	mRNA*	cDNA
Eukaryote	20μg	10μg	4μg
Class I Prokaryote	20μg	-	4μg
Class II and III Prokaryotes	30μg	-	8μg
*Please note that the NimbleGen procedure for sample processing does not include RNA amplification.

When I use NimbleGen full service option, what deliverables will I receive ?

NimbleGen delivers the following data files and software to full service customer:
- Microarray Design file containing probe sequences (ndf)
- Gene Description file (ngd)
- Raw Data files (calls and pair)
- Normalized Data files (RMA_calls and RMA_pair)
- ArrayStar v2.0 expression analysis software.

Array Delivery

Can I purchase NimbleGen arrays for use in my own lab or core facility?	Yes, all catalog and custom arrays are available for delivery.
Which gene expression designs are available for array delivery?	All catalog designs currently listed on NimbleGen website and any custom design are available for delivery.
What kits are available for use with NimbleGen microarrays?	NimbleGen offers Hybridization kits to facilitate efficient and specific hybridization to our expression arrays. See our Kits & Consumables web page for more details about these kits.
What resolution scanner do I need for scanning NimbleGen arrays?	A scanner with 5 μm resolution is required.
Can I scan NimbleGen arrays using my Agilent scanner?	Yes.
What software do I need to extract my data from a scanned image?	NimbleGen offers array delivery customers with NimbleScan data extraction and analysis software.
Does NimbleGen offer training for array delivery customers?	Yes, NimbleGen offers training at your lab or at our Madison, WI location. Please see our Training webpage for more details and a current schedule.
When I order microarrays from NimbleGen, what deliverables will I receive?	For microarray orders, NimbleGen delivers the following items to the customer: - NimbleGen arrays - Complete User Guide - Microarray Design file containing probe sequences (ndf) - Gene Description file (ngd) - ArrayStar expression analysis software (upon request)
How much sample do I need to run one microarray analysis in my lab?	When using NimbleGen-recommended protocol for cDNA synthesis and labeling, 10 μg total RNA, 1 μg mRNA, or 1 μg cDNA is required for each array.