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NimbleGen Sequence Capture - Product Info


Transform your sequencing project with this highly efficient and cost-effective method for capturing targeted regions of the genome.

Custom Array Delivery

NimbleGen Sequence Capture arrays enable you to produce targeted, sequencing-ready samples in your lab. Utilizing high-density, long-oligo NimbleGen arrays, only the human genomic regions you specify are hybridized and eluted, ready for subsequent amplification and sequencing. This array-based process offers significant speed and scalability advantages over current PCR-based methods for targeted enrichment.

Protocol
NimbleGen Sequence Capture Protocol
  1. The genomic DNA sample is fragmented by sonication or nebulization.
  2. The sample is hybridized to a NimbleGen Sequence Capture array.
  3. Unbound fragments are washed away.
  4. The target-enriched pool is eluted and LM-PCR amplified.
  5. The enriched sample is ready for high-throughput sequencing, such as with a 454 Genome Sequencer FLX instrument.
Advantages
  • High Performance: Capture up to 5Mb total regions on a single array with high coverage and specificity.
  • Design Expertise: Ensure the highest level of specificity and sensitivity with an empirically tested and validated capture design algorithm.
  • 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.
Array Design
  • 385,000 features per array
  • > 60bp probes
  • The latest genomic build from UCSC for human (HG18)
  • Optimized, empirically tested design algorithm (version 2.0) used for all designs
  • Only unique genomic regions tiled; repetitive regions are removed using our proprietary repeat-masking method
Performance
Optimized Design Algorithm (version 2.0)

Experimental Design

  • NimbleGen Sequence Capture 385K Arrays selected genomic regions as specified by the 1000 Genomes Consortium using: old design algorithm (version 1.0) OR optimized design algorithm (version 2.0)
  • Captured samples were sequenced using an entire PicoTiter plate on the GS FLX instrument using GS FLX standard chemistry (250bp reads; 100Mb raw sequence) OR GS FLX Titanium chemistry (400bp reads; 500Mb raw sequence)

Representative Results

  • Version 2.0 increases percentage of target bases covered (1x and 10x) compared to version 1.0 (24.3% increase for GS FLX Standard; 14.4% increase for GS FLX Titanium)
  • GS FLX Titanium chemistry increases percentage of target bases covered (1x and 10x) compared to GS FLX Standard (29.5% increase for version 1.0; 19.6% increase for version 2.0)
  • The powerful combination of the version 2.0 design algorithm and GS FLX chemistry resulted in 94.4% of all target bases having at least 10x fold coverage!

Comparison of Array Designs and Sequencing Chemistries

  Version 1.0 Version 2.0
Genome Sequencer (GS) Chemistry GS FLX Standard GS FLX Titanium GS FLX Standard GS FLX Titanium
Percentage of target bases covered 97.5% 98.2% 98.3% 98.7%
Percentage of target bases with at least 10x coverage 50.5% 80.0% 74.8% 94.4%

Exon Selection Example

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 Genome Sequencer 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

Figure 1. Sequence read map detail of ~190kb of chromosome 16 from three microarray direct selection replicate.
Contiguous Loci Example

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 Genome Sequencer 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 A

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

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 Genome Sequencer 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 1

Figure 3. The percentages of 454 Genome Sequencer FLX instrument reads that report variants, either from PCR or NimbleGen Sequence Capture, were plotted for each of the SNPs detected by both methods.
Workflows

Roche NimbleGen offers two workflow options for our NimbleGen Sequence Capture Arrays:

Delivery

  1. Roche NimbleGen certified trainers conduct Sequence Capture customer workshop on-site (3 days) to train researcher on protocols. Contact your local Sales representative for more details.
  2. Roche NimbleGen designs a NimbleGen Sequence Capture Array that targets regions specified by the researcher.
  3. Researcher orders NimbleGen Sequence Capture Arrays and gets set-up with essential equipment, reagents, and consumables.
  4. Researcher performs Sequence Capture experiment using our validated User’s Guide.
  5. Researcher sequences captured samples on 454 Genome Sequencer FLX (or micro-read technologies not supported by Roche NimbleGen).
  6. Learn more about Delivery...

Service

  1. Roche NimbleGen designs a NimbleGen Sequence Capture array that targets regions specified by the researcher.
  2. Researcher sends genomic DNA samples to the Roche NimbleGen Service Lab.
  3. Roche NimbleGen Service Lab captures and amplifies the targeted regions using NimbleGen Sequence Capture technology.
  4. Researcher receives sequencing-ready samples of enriched, amplified genomic fragments.
  5. Learn more about Service...
NimbleGen Sequence Capture Workflow

Figure 1: Sequence Capure Workflow Schematic
Click to see a more detailed image.

Literature

For a complete listing of literature covering all Roche NimbleGen products and services please visit our literature page.

General Documents

Delivery Workflow Documents

Service Workflow Documents

FAQ
Experimental Design
How much total sequence can I capture on your current NimbleGen Sequence Capture 385K custom arrays? The maximum amount of sequence that our current NimbleGen Sequence Capture 385K 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 through full service. 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 level of training does Roche NimbleGen provide for Sequence Capture Array delivery? Customers interested in being trained on the Sequence Capture protocol can take part in a 3 day on-site customer workshop conducted by our certified trainers. For more details contact your local Roche NimbleGen Sales representative.
Can I perform Sequence Capture on organisms other than human using Array Delivery? Performing Sequence Capture on other organisms can be done by ordering Sequence Capture arrays for delivery. However, Roche NimbleGen does not support the use of these arrays for Sequence Capture. QC tests must be developed by the researcher to ensure a successful capture.
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.
Can I pool 2 DNA samples for targeted re-sequencing? Yes, but you will need to sequence deeper to indentify SNPs and indels as more sequences are pooled. Resolving the individual captured samples by sequencing has not yet been developed.
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 build of human is Roche NimbleGen using to design a 385K custom array? We are using the latest genomic build for human (HG18).
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 genome.
What genomic database is acceptable for submitting my design coordinates? At this time we are only accepting genomic coordinates for custom design using the UCSC database.
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.
Can I re-use NimbleGen Sequence Capture arrays? No, we do not recommend re-use at this time.
Sample Requirements
What are the sample requirements for a NimbleGen Sequence Capture full service experiment? We require at least 21μg human genomic DNA at a concentration of 250-500ng/μl per array. 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
If I am planning on using 454’s sequencing service to sequence my captured DNA (using Full-Service option), can my samples be sent directly to 454? Yes, Roche NimbleGen and 454 Life Sciences have a combined service where your captured samples are sent directly from our Service Lab to 454’s Service Lab, without you ever having captured samples back in your possession. Contact 454 (www.454.com) Life Sciences if you are interested in this combined service option.
After the Roche NimbleGen Service Lab captures my desired sequences, what do I get back? You will receive 10μg 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 and a user's guide that describes how to sequence the captured DNA using the 454 Genome Sequencer FLX instrument. GFF files can be anaylyzed with Roche NimbleGen SignalMap software. A free, 30-day demo version of SignalMap software is available for download.
Will the Array Delivery User’s Guide that is optimized for human Sequence Capture be publically available for capture of other organisms (for empirical protocol development)? Yes, the User’s Guide is available for download from the Sequence Capture homepage.
Downstream Applications
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 Genome Sequencer 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 arrays. In the future, using NimbleGen 2.1M format arrays in conjunction with 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 400bp because long reads enable comprehensive variant detection. To supply our customers with an affordable, high-quality solution, we have been working closely with 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 Genome Sequencer FLX instrument. The 454 Genome Sequencer FLX instrument with FLX Titanium Kits delivers read lengths of 400bp (500MB raw sequence per PTP) 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 advantage of 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
Will Roche NimbleGen offer NimbleGen Sequence Capture products on the HD2 (2.1 million probes) platform? Yes, we are planning to release NimbleGen HD2 arrays as part of the NimbleGen Sequence Capture product offering in early 2009. With NimbleGen HD2 arrays, you can capture up to 30Mb of sequence, compared to 5Mb with the current 385K arrays.
What NimbleGen Sequence Capture products will be offered in the future? We will launch Sequence Capture HD2 Arrays - capable of capturing the human exome on a single slide - in early 2009. This array tiles through approximately 150,000 of the best annotated CCDS exons.
Can I use NimbleGen Sequence Capture 385K Custom Delivery Arrays for DNA methylation analysis? At this time, Roche NimbleGen does not support the use of Sequence Capture products with bisulfite techniques for analyzing single nucleotide DNA methylation status. However, we are developing and optimizing products and kits that will allow for DNA methylation analysis using Sequence Capture in late 2009.
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