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.

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 GS 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.

• Highly optimized workflow reduces hands-on time, lowers sample requirements from 20µg to 5µg, and increases the usable sequence by 10%.
• The unique 400bp long reads from the GS FLX Titanium Series enable easy detection of small indels, improve coverage in repetitive regions, and provide haplotype information.
• Dedicated GS Reference Mapper software reports variant locations, amino acid changes in coding regions, and known SNP information. In addition, the GS Mapper software generates capture performance metrics, such as percentage reads in target regions.
• Find out more about the Genome Sequencer Instrument...

• High Performance: Capture up to 30Mb total regions on a single 2.1M array and up to 5Mb on a single 385K 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.

• 2.1M and 385,000 features per array
• > 60bp probes
• The latest genomic build from UCSC for human (HG18). 2.1M Human Exome Arrays (HG18) designed using the April 30th, 2008 build from the CCDS database.
• 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
• Click here to learn how to generate a target sequence list of coordinates for a custom design using UCSC Genome Browser

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; 400Mb 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%

Exonic Enrichment

Experimental Design NimbleGen 385K arrays targeted 6,726 cancer exons (500bp minimum size) Probes tiled regions every 10bp Probe lengths ranged from 60 - 90bp 4.55Mb of total sequence (0.15% of the genome) targeted Selected from Burkitt’s Lymphoma cell line DNA Three replicate microarray selections performed 454 sequencing was performed using a full PTP on each replicate using the Genome Sequencer FLX and Standard Series Kits (250bp reads) Representative Results Targeted genomic bases 4,550,667 Total 454 sequencing reads 304,250 Number of reads in target regions 239,139 Percent of reads in target regions 78.6 Targeted bases covered 4,356,030 Percent target bases covered 95.7 Average fold coverage 10.1 Median fold coverage 9 Figure 1 Figure 1. Exonic Enrichment with NimbleGen Sequence Capture Arrays Shown is a small sample of a parallel enrichment of 6,700 exonic target sequences, performed using NimbleGen Sequence Capture 385K arrays from full complexity human genomic DNA. The plotted regions are from chromosome 7. In Track A, the array-targeted regions are indicated in green. In Track B, observed sequencing fold coverage is indicated in red. In Track C, individual sequencing reads, sorted by size, are shown in blue. For this data set, the median sequencing coverage was 9-fold using a single NimbleGen Sequence Capture 385K array and a single, full plate Genome Sequencer FLX sequencing run using Standard Series Kits. In the zoom view, the level of target accuracy is illustrated with more than 78% of reads falling within targeted regions.

Contiguous Locus

Experimental Design 3Mb (primary region) contiguous region on human chromosome 11p12 involved in Type II diabetes susceptibility was targeted using NimbleGen 385K arrays against 2.1Mb (capture region) Repeat regions were masked from selection Selected from HapMap sample 454 Sequencing was performed using a full PTP on the Genome Sequencer FLX instrument and Standard Series Kits (250bp reads) Representative Results Targeted genomic bases 2,113,165 Total 454 sequencing reads 365,261 Number of reads in target regions 295,628 Percent of reads in target regions 80.9 Targeted bases covered 2,095,716 Percent target bases covered 99.2 Average fold coverage 28.7 Median fold coverage 28 Figure 2 Figure 2. Enrichment of a Contiguous Genomic Region using NimbleGen Sequence Capture Arrays Shown is a plot of a portion of the sequencing reads obtained from a 2.1Mb genomic region from human chromosome 11p12 enriched from full complexity genomic DNA. In Track A the regions targeted for capture are indicated in green. In Track B, the observed fold coverage for sequencing in the array-targeted region using a Genome Sequencer FLX Instrument and Standard Series Kits is shown in red. Track C is a plot of the entire set of individual sequencing reads, sorted by read length from less than 30 bases to in excess of 300, plotted in blue. The zoom view shows the right edge of the targeted region. The observed sequencing reads show high specificity to the targeted region.

Accurate Detection of SNPs

Experiment Sequencing Run Total Reads Total Bases On-Target Reads Median Coverage Target Bases with 10+ Coverage Target Bases with 1+ Coverage No. of Known SNPs in Target Region No. of Known SNPs Called Correcetly SNP Detection Rate 250kb-1 1/8 PTP 70190 27646394 75.2% 85 97.3% 98.6% 273 266 97.4% 250kb-2 1/8 PTP 86814 33422118 67.2% 92 97.4% 98.7% 273 265 97.1% 250kb-3 1/8 PTP 93322 34246259 83.4% 116 97.6% 98.9% 273 268 98.2% 250kb-4 1/8 PTP 67339 26271625 45.9% 48 95.6% 97.5% 273 265 97.1% 1Mb-1 1/4 PTP 140374 55453593 87.3% 49 92.8% 96.9% 832 803 96.5% 1Mb-2 1/4 PTP 123957 49429493 84.7% 42 92.7% 96.9% 832 797 95.8% 1Mb-3 1/4 PTP 163480 64046746 91.3% 60 94.5% 97.2% 832 800 96.2% 1Mb-4 1/4 PTP 228493 89501377 71.5% 65 94.5% 97.3% 832 803 96.5% Table 1. NimbleGen 454 Optimized Sequence Capture 385K arrays were used to capture both a 250kb contiguous region, and a 1Mb contiguous region in the human genome from a HapMap DNA sample. Four independent experiments were performed by external evaluators for each design. Sequencing data were generated using Genome Sequencer FLX System and GS XLR70 Sequencing Kit on PicoTiterPlate (PTP) device. The PTP is divided into 8 regions by a gasket, and each sample from a 250kb capture was sequenced by one of the 8 regions (1/8 PTP), while each sample from 1Mb capture is sequenced by two of the 8 regions (two 1/8 PTP, or roughly equal to one region from a 4 region gasket{1/4 PTP}). Data were analyzed using the GS Reference Mapper software, and SNP calls were compared to known HapMap SNPs.

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 OR the customer selects a catalog design (2.1M Human Exome). Download the 2.1M Human Exome Annotation Files to see if your gene(s) of interest are on the Exon array.
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 OR the customer selects a catalog design (2.1M Human Exome).
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...

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

General Documents

• Sequence Capture Microarrays and Services
  Brochure (PDF Format 2MB)
• Sequence Capture Custom Designs: Guide to Submitting Your Target Sequence
  User’s Guide (PDF Format 906KB)

SeqCap EZ Exome Documents

• SeqCap EZ Exome Library & 454 GS FLX Titanium Series
  Sales Flyer (PDF Format 1.5MB)
• SeqCap EZ Exome Library LR
  User’s Guide (PDF Format 712KB)
• SeqCap EZ Exome for Short Read Sequencing
  Sales Flyer (PDF Format 1.1MB)
• SeqCap EZ Exome Library SR
  User’s Guide (PDF Format 866KB)

Delivery Workflow Documents

• NimbleGen 454 Optimized Sequence Capture 385K Arrays
  Sales Flyer (PDF Format 473KB)
• NimbleGen Sequence Capture 2.1M Human Exome Array
  Sales Flyer (PDF Format 977KB)
• NimbleGen Sequence Capture 385K Version 2.0 Arrays: Custom Human Arrays for Delivery
  Sales Flyer (PDF Format 309KB)
• NimbleGen Sequence Capture 2.1M and 385K Arrays: Equipment and Reagent Requirements
  Sales Flyer (PDF Format 247KB)
• NimbleGen Arrays User’s Guide: 454 Optimized Sequence Capture Array Delivery (Version 1.1)
  User’s Guide (PDF Format 1.3MB)
• NimbleGen Arrays User’s Guide: Sequence Capture Array Delivery (Version 3.2)
  User’s Guide (PDF Format 3.4MB)
• 2.1M Human Exome Annotation Files
  Annotation Files (ZIP Format 8.7MB)
  What files are included in this download?

Service Workflow Documents

• NimbleGen Sequence Capture - Full Service Solution
  Sales Flyer (PDF Format 275KB)
• NimbleGen Services User’s Guide: Sequence Capture Service (Version 3.0)
  User’s Guide (PDF Format 2.2MB)
• NimbleGen Sequence Capture 2.1M Human Exome Array
  Sales Flyer (PDF Format 977KB)

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

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Experimental Design

Can I send my gDNA samples to a service provider for genomic enrichment in my area? Roche NimbleGen is currently establishing certified service providers (CSP) globally. Please click here to check for available CSPs in your area. How much total sequence can I capture on your NimbleGen Sequence Capture 2.1M and 385K custom arrays? The maximum amount of sequence that our current NimbleGen Sequence Capture 2.1M arrays can capture is 30Mb. Our 385K arrays can capture up to 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. 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 generate a target sequence list of coordinates for a custom design? Roche NimbleGen provides a guide for generating and submitting target sequences for your custom design. Click here to find out more. 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 human genome build is Roche NimbleGen using to design a Sequence Capture custom array? We are using the HG18 build. 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 385K or HD2 Exome 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

After the Roche NimbleGen Service Lab captures my desired sequences, what do I get back? You will receive 6μg 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 (.gff and .bed), a coverage summary.txt file showing coverage of the design by probes, and a User's Guide that describes how to sequence the captured DNA using the 454 Genome Sequencer FLX Instrument and either GS FLX Standard series kits or GS FLX Titanium Series kits. 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 (Version 3.2) 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 30Mb using 385K and 2.1M arrays. 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). 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 GS 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

Is Roche NimbleGen optimizing their Sequence Capture technology with any of the next-generation sequencing platforms? Yes, we are currently developing 385K Sequence Capture products that use a protocol optimized for the 454 GS FLX Instrument using GS FLX Titanium Series Kits. This provides a seamless transition from capture into sequencing since no library preparation needs to be performed after array capture. Captured fragments are ready to go into the emPCR Amplification step.

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