Overview of working with Llibraries and library sets

In the SureSelect Target Enrichment and SureSelect RNA Enrichment application types in eArray, a library is a collection of biotinylated RNA oligonucleotide baits that you can use to isolate specific RNAs or genomic DNA fragments for sequencing. You can use eArray to find, upload, create, and organize the nucleotide sequences of the baits that are represented in custom libraries. Once you create a library, you can submit it to Agilent Manufacturing, which makes it available for price quotes and orders. You can request a quote for the library from within eArray, and you can also place an order for the library through the Agilent Online Store or your Agilent representative.

Libraries contain one or more bait groups, and each bait group contains individual baits. eArray supports several types of libraries. See these topics:

Overview of SureSelect enrichment libraries

SLearn about SureSelect Target Enrichment libraries

SLearn about SureSelect RNA Enrichment libraries

BOverview of working with baits

BOverview of working with bait groups

For the SureSelect Target Enrichment application type, eArray creates library sets automatically when you design a library that has more baits than can be accommodated by a single library. See Library sets, below.

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 that 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 cover specified genomic regions or transcript sequences of a given species. See Bait Tiling wizard (SureSelect Target Enrichment) and Bait Tiling wizard (SureSelect RNA Enrichment).You can optimize the bait creation process for your specific sequencer and sequencing protocol, and also customize the tiling density. In addition, you can enter regions to avoid.
Combine Base and Supplemental Libraries – Creates a library that contains a selected Agilent Catalog library (the base library) and an additional library (the supplemental library). This process lets you add baits for genomic regions or transcripts that may not be included in the catalog library. Before you use this wizard, you must first create or find a custom supplemental library that contains the desired additional baits. You then include the base and supplemental libraries in the combined library. For the SureSelect RNA Enrichment application type, the supplemental library can be any existing custom SureSelect RNA Enrichment library. For the SureSelect Target Enrichment application type, you must use the Bait Tiling wizard to create a supplemental library that is matched to the desired base 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, request a quote for it, and order it from the Agilent Online Store through eArray. 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, which lets you sequence multiple input fragments in a single sequencing reaction. Multiplexing is always enabled for certain libraries, and is available as an option for others. You select multiplexing when you request a quote for a library or order it from the Agilent Online Store through eArray.

Note: If you use the eArray_XD program in Agilent Genomic Workbench to submit SureSelect Target Enrichment libraries to Agilent, these libraries are also available in your account on the eArray Web site. Some restrictions apply. See Search for eArray_XD libraries.

Library sets (SureSelect Target Enrichment application type only)

As part of the library manufacturing process, Agilent uses its proprietary SurePrint technology to print oligonucleotides onto glass slides. One glass slide accommodates a single 1 X 55K library, which can contain up to 57,750 baits.

If you create a library that contains more than 57,750 baits, eArray automatically creates a library set, which distributes the requested baits evenly among multiple libraries (and thus multiple slides). If you subsequently decrease the number of baits to 57,750 or fewer, eArray re-accommodates the baits in a single library on one slide. eArray can create library sets that contain up to five individual libraries.

When you purchase a SureSelect Target Enrichment kit that is based on a library set, the baits from the individual libraries in the set are supplied in separate tubes. For example, if you create a library that contains 150,000 baits, eArray creates a library set that contains three equal-sized libraries. The kit that is based on this library set contains three tubes of baits, and each tube contains 50,000 baits.

To take action on library sets, you use the same commands that you use for individual libraries. For example, you view or edit a library set the same way that you can view or edit a library. Library sets are handled as a single entity—you cannot separately order or work with the individual libraries in a library set. Because eArray automatically creates a library set when one as needed, you can design very large libraries as easily as you design smaller ones. You can add bait groups to a library set as if you were adding them to a single library. eArray creates the appropriately-sized library set, and evenly distributes baits to each of the libraries in the set automatically.

Note:

• You cannot separately work with or order the individual libraries in a library set.

• A library set can contain up to five individual libraries. Therefore, a library set can accommodate a maximum of 288,750 baits, including all Agilent quality control baits.

• Library sets are automatically enabled or disabled by the eArray system based on the total number of requested baits. You cannot manually enable or disable library sets.

• Library set support for specialized types of libraries, such as supplemental or combined libraries, is not available.

• When you design an individual custom bait library, it has a library type of Normal. If you add baits to the library, and eArray enables a library set, the library type changes to Custom Library Set.

• eArray treats library sets as a distinct type of library with a library type of Custom Library Set. Thus, when you search for libraries that have a library type of Normal, no library sets are returned. To retrieve library sets, you must select a library type of All or Custom Library Sets. Similarly, when you browse the custom libraries in your eArray account, if you limit the list of libraries to those with a type of Normal, no library sets appear. To include library sets in the browse list, you must either not filter the browse list, or filter the browse list to contain libraries that have a library type of Custom Library Sets.

• Library set support is only available for the SureSelect Target Enrichment application type.

Status of SureSelect enrichment libraries

The status of a library reflects its progress through the library creation process. Status designations follow a defined order:

Draft >>> Review (optional) >>> Locking (collaborations only) >>> Complete >>> Submitted

In some cases, eArray assigns the status, and in others, the owner of the library advances the library to the next status. This is a one-way process — once you advance a library to the next status, you cannot go back.

The actions that you can take on a library depend on the current status of the library, the privileges assigned to you by your workgroup administrator, and if you are outside or inside of a collaboration. The table below describes each of the statuses.

Status	Description	Notes
Draft (not available inside a collaboration)	You are at the beginning of the library design process. eArray assigns this status to your library when you first create it.	Only the owner of the library can edit it. The owner of the library can change the status of the library to Review or Complete. Only the owner of the library can delete it. The library cannot be submitted.
Review	This is an optional status that the owner of the library can set. It lets others make changes to the library. See Review library.	Users with access privileges can review the library, make changes, and save new versions of it. A .tdt file is dynamically generated every time a reviewer saves a new version. Only the owner of the library can delete it. Anyone who has access privileges can download the library (.tdt file). The library cannot be submitted.
Locking (Available only in a collaboration)	You, as a lead in a collaboration, set this status when you want to lock the library to prevent additional edits	Only a collaboration lead can lock a library. The library has a status of Locking until all leads in the collaboration approve Locking. The status of the library then becomes Complete.
Complete	In your individual user workspace, you, as the owner of the library, set this status when you and any reviewers have made all desired edits to it. In a collaboration, eArray assigns this status when the last lead gives approval to lock the library.	ELID number is assigned to the library. eArray generates all library files. Library cannot be edited (not even by its owner). Owner can submit the library to Agilent Manufacturing. Anyone who has access privileges can download the library design files and the tab-delimited text (*.tdt) file. The library can be deleted by its owner. The owner of the library can change the control type of bait groups in the library.
Submitted	eArray sets this status after you submit the library to Agilent Manufacturing.	Only the owner of the library can submit it. In a collaboration, all leads must approve the submission. Only libraries with a status of Complete can be submitted. After the library is submitted, eArray submits the library design file to Agilent Manufacturing. A library cannot be edited or deleted after it is submitted. The owner of the library can change the control type of bait groups in the library. Anyone with access to the library can request a quote for it. If you have previously purchased products from Agilent, you can or Order the library from the Agilent Online Store through eArray. Subscription to library update notification is available.

Guidance from Agilent

Agilent offers the following recommendations for 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 each 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.

Agilent offers the following recommendations for the design of SureSelect RNA Enrichment libraries:

The cumulative base coverage should be from 0.2 to 3.2 Mb.
Use a tiling frequency of 2x.