In the Agilent target enrichment protocol, a library is a collection of biotinylated RNA oligonucleotide baits that you use to isolate specific genomic DNA fragments for sequencing. You can use eArray to find, upload, create, and organize the nucleotide sequences represented in custom libraries. Once you create a library, you can submit it to Agilent Manufacturing, who then makes it available. You request a quote for the library from within eArray, and later place an order through your Agilent representative.
Libraries contain one or more bait groups, and each bait group contains individual baits. See Target enrichment and eArray, Baits, and Bait groups.
One of the easiest ways to create a library is to use one of the built-in Library Wizards that are available in eArray, which take you step-by-step through available library creation processes. Each wizard concludes with an opportunity to submit the library to Agilent Manufacturing. These Library Wizards are available:
Create Library from Bait Upload – Creates a library of baits based on baits in a file you upload.
Create Library from Existing Bait Group(s) – Creates a library of baits based on a search for bait groups. The bait groups can be from the Agilent Catalog, or bait groups in any folder within the eArray system to which you have access.
Create Library by Bait Tiling – Creates a library of baits that evenly span specified genomic regions of your species of interest. You can optimize the bait creation process for your specific sequencer and sequencing protocol, and also customize the tiling density and the length of baits. In addition, you can specify the specific desired genomic regions, as well as regions to avoid.
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.
Agilent offers the following recommendations regarding the design of SureSelect Target Enrichment Libraries:
Avoid repeat regions in bait design with fairly high stringency (at most 20 bp overlap).
Follow the "Optimized" bait design strategy (when you set up Bait Tiling, mark Use Optimized Parameters). These parameters have been found to work well under standard conditions and are optimized for a given 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.