Ways to create SureSelect libraries |
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Note: 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.
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 from Bait Upload – Use this wizard to create a library based on a file of bait sequences that you upload.
Create Library from Existing Bait Groups – Use this wizard to design a library that includes existing collection(s) (groups) of baits. You can include your own bait groups if they are in your user account on eArray, and you can also include bait groups from the Agilent Catalog.
Create Library by Bail Tiling (Target Enrichment) or Create Library by Bail Tiling (RNA Enrichment) – Use one of these wizards to design a library based on oligonucleotides that cover specific regions of the genome of interest at even intervals.
Combine Libraries (Target Enrichment) – Creates a combined library that contains a selected Agilent catalog library (the base library), and a custom library (the additional library). This process lets you add baits for regions that are not included in the catalog library.
eArray also allows you to manually create a library from bait group search or browse results.
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.
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.