Target enrichment, also known as genome partitioning, is a method that isolates specific fragments of genomic DNA for sequencing. You use a library of complementary oligonucleotide "baits" to harvest fragments of interest (target DNA). The target DNA hybridizes well with the baits, but other DNA does not, which forms the basis of a powerful selection method that enables you to focus your sequencing efforts.
When you order a SureSelect Target Enrichment library from Agilent, the final product that you receive is a kit that contains a set of biotinylated RNA oligonucleotides. However, as part of the library manufacturing process, Agilent first creates DNA oligonucleotides, and then later transcribes them into RNA. Thus, when you create a library in eArray, you specify bait sequences in terms of DNA bases (A, C, G, T).
With eArray, you can access existing baits and bait libraries or create custom libraries of baits. A bait library is a collection of oligonucleotides in a tube, not a microarray, but you can leverage the well-developed eArray microarray creation infrastructure to easily design and obtain the libraries that you need for your research. To use eArray to create bait libraries, you must be an eArray registered user. When you log in to eArray, you set the Application Type to SureSelect Target Enrichment, and the relevant tabs and commands appear. When you become a registered user, you also gain access to eArray's large, robust set of microarray creation tools.
In eArray, bait libraries contain one or more bait groups, and each bait group contains individual baits:
eArray stores individual baits as well as bait groups as separate entities, so you can use them as building blocks to make many different libraries.
For individual baits, you can use existing baits within eArray. To put your own baits on eArray, you can upload them, and you can also use Bait Tiling, a method that creates baits based on spanning selected regions of a genome of interest at even intervals. eArray supports a bait length of 120 nucleotides for all users. To enable bait lengths of 90, 150, and/or 170 nucleotides for your workgroup, contact Agilent Technical Support.
To organize baits into bait groups, you can select baits based on a Search Your Baits search. You can also create a bait group that contains baits that you upload or those that you create with the Bait Tiling tool. Baits and bait groups are separate entities—you can use an individual bait in any number of bait groups.
One of the easiest ways to design bait libraries is to use one of eArray's built-in Library Wizards. Each wizard leads you step-by-step through the library creation process. As you complete a wizard, you can submit your newly-designed library to Agilent Manufacturing. To obtain the library, you request a quote online through eArray, and then place an order through your Agilent representative. eArray has several Library Wizards:
Create Library by Bait Tiling – Use this wizard to design a library based on oligonucleotides that span specific regions of the genome of interest at even intervals.
Create Library from Existing Bait Groups – Use this wizard to design a library using existing collection(s) (groups) of baits. You can include your own bait groups if they are in the eArray system, and you can also include bait groups from the Agilent Catalog.
Create Library from Bait Upload – Use this wizard to create a library based on a file of bait sequences that you upload.
Create Supplemental Library by Bait Tiling – Creates a library of baits that are tiled to genomic regions that you enter. The system sets the Bait Tiling parameters based on the ones that are defined for an existing base library that you select. In this way, you can create a library of additional baits that you can add to that base library when you create a combined library.
Create Combined Library – Creates a library that contains a selected base library and a supplemental library. This process is available for certain Agilent Catalog libraries, and it lets you add baits for regions that are not included in the catalog library. Before you use this wizard, you must first create a supplemental library based on the selected Agilent Catalog library.
eArray also gives you other options for libraries:
You can create a custom library manually from bait group search or browse results.
If your desired library already exists within eArray, either in the Agilent Catalog or in a folder to which you have access, you can search or browse for it. You can then submit it to Agilent Manufacturing and request a quote for it. Search and browse results also give you several other commands that you can use to manage libraries. See Actions you can take on libraries.
You can create a library to be used for multiplexing. Multiplexing is always enabled for certain smaller libraries, and is available as an option for other libraries. You select multiplexing when you request a quote for a library.
As you create libraries, they go through several status changes. The status designations for libraries parallel those for microarray designs. For a description of these designations, see Overview of microarray design/set status.
If you require greater flexibility, or want to focus your attention on one aspect of the process at a time, eArray lets you do this, as well. See the following topics for more details:
Baits
Bait groups
Libraries
Agilent provides these recommendations for the creation of custom SureSelect Target Enrichment Bait Libraries:
When you use Bait Tiling, avoid repeat regions with fairly high stringency (at most 20 bp overlap).
Use the "Optimized" Bait Tiling strategy. To do so, mark the Use Optimized Parameters check box when you set up a Bait Tiling job. These parameters have been found to work well under standard conditions and are optimized for each available sequencing technology.
The SureSelect Target Enrichment protocol is optimized for capture regions of cumulative size greater than 1 Mb. Therefore, optimal results will be obtained with 1Mb+ captures.
Additional recommendations from Agilent can be found in the topic Bait tiling. Also, you may find the Agilent tutorials on the SureSelect Target Enrichment application type to be useful. To view these tutorials, go to the eArray Login page. Under Additional Information, click SureSelect Target Enrichment Tutorial with Wizard or SureSelect Target Enrichment Tutorial without Wizard.
eArray makes it easy for you to work with others down the hall, or across the world. You can create collaborations with others, then work together to construct and submit bait libraries of mutual interest. You create a special collaboration workspace that has many of the same tools as your individual user workspace. See the following topics for more details:
Collaborations
What are the roles in a collaboration
Create a collaboration