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NimbleGen Sequence Capture - Delivery Workflow


Sequence Capture Delivery Workflow
The Delivery Process
  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).
Designs

Array Design Approval Process

There are two tracks in the design files: primary target region and capture target.

  1. NimbleGen's SignalMap software allows you to visually inspect the genomic feature format (GFF) file of your custom array design. A free, 30-day demo version of SignalMap is available for download.
  2. The internet-based UCSC Genome browser can be used to visually inspect the BED file of your custom array design.
  3. Inspect the coverage of your custom array design by opening the coverage summary file (.txt) using WordPad or Notepad.

2.1M Human Exome

  • Uses optimized design algorithm to target ~180,000 human coding exons and ~550 miRNA exons from the CCDS database (April 30, 2008)
  • Capture whole human exome on a single array in as little as 1 week
Equipment
NimbleGen Hybridization System
Spacing Image 4-bay Hybrization System
  • Hybridize your fragmented , linker-ligated DNA to Sequence Capture Array
  • Available in a 4-bay (shown) or 12-bay model. See our Instruments page for more information.
NimbleGen Elution System
Spacing Image NimbleGen Elution System
  • Elute your captured fragments from the array in a matter of minutes!
  • See our Instruments page for more information.
Roche LightCycler® Real-Time PCR System
Spacing Image Roche LightCyler 2.0
  • Determine the success of your Sequence Capture experiment by measuring fold-enrichment across 4 control loci with qPCR
  • Cost-effective method for determination of capture sample quality PRIOR to next-generation sequencing.
  • Find out more...
454 Genome Sequencer FLX System and GS FLX Titanium Series Kits
Spacing Image 454 Genome Sequencer FLX Instrument
  • Captured samples can be fed directly into the 454 GS FLX Titanium Shot-gun library preparation protocol for addition of sequencing adapters.
  • Sequence your captured samples on the 454 GS FLX to generate high-confidence sequencing data using up to ~1,000,000 reads for roughly 500Mb of raw sequence data per picotiter plate.
  • 400bp reads enable haplotyping of 400bp and identification of insertions/deletions from single to tens of base pairs.
  • Find out more...
Kits

Use the NimbleGen Sequence Capture Hybridization Kit to perform up to 30 hybridizations (2.1M) or 60 hybridizations (385K) of fragmented gDNA libraries to a NimbleGen Sequence Capture array.

The NimbleGen Sequence Capture Wash and Elution Kit contains optimized buffers to ensure efficient washing of NimbleGen Sequence Capture arrays after hybridization. You can perform 15 elutions on 2.1M and 385K arrays with the components in one kit.

Catalog Number Name Kit
Capacity		 Kit
Components
05340721001 NimbleGen Sequence Capture Hybridization Kit See Below See Below
Format Number of Hybridizations Number of Slides
2.1M 30 30
385K 60 60
Component Storage Temperature 1 Quantity Volume per Tube
2X SC Hybridization Buffer 4°C 1 600µl
SC Hybridization Component A 4°C 1 250µl
1 The kit is shipped on blue ice (-20 ºC). Upon receipt of the kit, remove the components and store them at the recommended storage temperatures noted in the table above.
05340730001 NimbleGen Sequence Capture Wash and Elution Kit See Below See Below
Format Number of Washes/Elutions Number of Slides
2.1M 15 15
385K 15 15
Component Storage Temperature Quantity Volume per Tube
2X Stringent Wash Buffer Room Temperature 1 500ml
10X SC Wash Buffer I Room Temperature 1 50ml
10X SC Wash Buffer II Room Temperature 1 250ml
10X SC Wash Buffer III Room Temperature 1 50ml
Elution Reagent Room Temperature 2 25ml

For a complete listing of Roche NimbleGen Kits please visit our Kits & Consumables page.

Deliverables
Literature

General Documents

SeqCap EZ Exome Documents

Delivery Workflow Documents

Service Workflow Documents

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

FAQ
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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Product Interest * SeqCap EZ Exome Library LR (Long Read)
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Sequence Capture Human Custom 2.1M Array
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Sequence Capture 385K Array
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