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Transform your sequencing project with this highly efficient and cost-effective method for capturing targeted regions of the genome.
NimbleGen Sequence Capture technology is a revolutionary process that enables the selective enrichment of up to 5Mb of selected genomic regions from full-complexity human or mouse genomic DNA in a single enrichment step. The process utilizes high-density, long-oligo NimbleGen arrays for the targeted hybridization and elution of only those genome regions you specify. The method is applicable to large contiguous or noncontiguous genomic regions, whole exomes, or any other targeted regions you want to sequence.
NimbleGen Sequence Capture Protocol 
- The genomic DNA sample is fragmented.
- The sample is hybridized to a custom NimbleGen Sequence Capture array.
- Unbound fragments are removed.
- The target-enriched pool is eluted and amplified.
- The enriched sample is ready for high-throughput sequencing, such as with a 454 GS FLX instrument.
NimbleGen Sequence Capture Advantages
- High Performance: Capture up to 5Mb total regions on a single array with high coverage and specificity.
- Embedded Quality Controls: NimbleGen Sequence Capture arrays incorporate built-in control probes to ensure system performance.
- Maximum Flexibility: Tailor the array design to capture your genomic regions or thousands of exons in parallel.
- Substantial Savings: Save time and cost compared to PCR-based methods.
NimbleGen Sequence Capture Microarray Designs
- 385,000 features per array
- Long oligo probes
- The latest genomic builds from UCSC for human (HG18) and mouse (MM9)
- Only unique genomic regions tiled; repetitive regions are removed using our proprietary repeat-masking method
NimbleGen Sequence Capture Performance
| Exon Selection Example |
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Experimental Design
- Microarrays selected 6,726 cancer exons (500bp minimum size)
- Microarray probes tiled regions every 10bp
- Probe lengths ranged from 60 - 90bp
- 5Mb of total sequence (0.15% of the genome) selected
- Selected from Burkitt’s Lymphoma cell line DNA (whole-genome amplified)
- Three replicate microarray selections performed
- 454 Genome Sequencer FLX sequencing performed on each replicate
Representative Results
- Three 454 GS FLX runs produced 63Mb, 115Mb, and 93Mb each
- 75%, 65%, and 76% of reads mapped to target regions
- 96%, 93%, and 95% of target regions had sequence coverage
- Median per-base sequence coverage was 5-, 7-, and 7-fold, respectively
Figure 1. Sequence read map detail of ~190kb of chromosome 16 from three microarray direct selection replicate. |
| Contiguous Loci Example |
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Experimental Design
- Five regions of increasing size surrounding the BRCA1 gene locus on chromosome 17 were selected using different microarray designs
- Repeat regions were masked from selection
- Selected from Burkitt’s Lymphoma cell line DNA (whole-genome amplified)
- 454 GS FLX sequencing performed on each captured region
Representative Results
| Tiling Size |
Avg. Selection
Probe Tiling Density* |
FLX–Yield |
Median Fold Coverage
of Unique Portion of Region |
| 200kb |
1bp |
102Mb |
79 |
| 500kb |
1bp |
85Mb |
93 |
| 1,000kb |
2bp |
96.7Mb |
38 |
| 2000kb |
3bp |
112.6Mb |
37 |
| 5,000kb |
7bp |
140Mb |
18 |
| * Probe Tiling Density refers to the average distance (in bp) between adjacent probes. |
Figure 2. Sequence read map detail of ~2,000 bases of chromosome 17 from a microarray selection of a 2Mb contiguous region that contains the BRCA1 gene. |
| No Bias Against Novel Variants Example |
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Experimental Design
- Compare performance of NimbleGen Sequence Capture technology vs. long range PCR
- A 200kb region surrounding the EFGR gene was captured using a NimbleGen Sequence Capture microarray
- A 70kb region (from the 200kb) region was amplified using long range PCR
- DNA samples derived from target regions using both methods were sequenced separately using the 454 GS FLX instrument
- SNP discovery was performed on both data sets
Representative Results
- Almost all the variants were captured with the same fidelity as PCR
- In the 70kb region targeted by both methods, 98 SNPs were identified by both
- Among rare variants, 9 and 5 were identified by NimbleGen Sequence Capture technology and long range PCR, respectively
- In repeat regions, 22 were detected only by long range PCR because no probes were designed to repetitive regions on the array
Figure 3. The percentages of 454 GS FLX instrument reads that report variants, either from PCR or NimbleGen Sequence Capture, were plotted for each of the SNPs detected by both methods. |
NimbleGen Sequence Capture Full Service
Array Design Approval Process
There are two tracks in the design files: primary target region and capture target.
- NimbleGen's SignalMap software allows you to visually inspect the genomic feature format (GFF) file of your custom array design.
- The internet-based UCSC Genome browser can be used to visually inspect the BED file of your custom array design.
The Service Process
- Roche NimbleGen designs a NimbleGen Sequence Capture array that targets regions specified by the researcher.
- Researcher sends genomic DNA samples to the Roche NimbleGen Service Lab.
- Roche NimbleGen Service Lab captures and amplifies the targeted regions using NimbleGen Sequence Capture technology.
- Researcher receives sequencing-ready samples of enriched, amplified genomic regions.
Sample Requirements
| Sample Required |
21μg each sample |
| Sample Concentration |
250-500ng/μl |
| A260/A280 ratio |
≥ 1.8 |
| A260/A230 ratio |
≥ 1.9 |
Quality Control
- Proprietary method for determining effectiveness of the sequence capture prior to next-generation sequencing
- Measurement of purity and yield of captured DNA
Deliverables
The deliverables provided with NimbleGen's Sequence Capture service includes:
- Yield and quality control report
- Target regions GFF and BED file
- 10μg captured DNA per submitted sample
- SignalMap GFF visualization software
- User’s guide, including how to perform sequencing with the 454 GS FLX instrument
FAQ
| Hide All Topics Show All Topics |
| 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. |
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| 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.
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| 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. |
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| 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. |
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| 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.
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| 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. |
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| 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. |
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Request a Quote for Sequence Capture Service
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