Create library by Bait Tiling (wizard) in SureSelect Target Enrichment

Parameter

Instructions/Details

Tiling Job Parameters

Job Name

Type a name that will later let you identify this Bait Tiling job.

Species

Select the desired species. This list contains the names of all species currently supported in eArray for Bait Tiling.

Sequencing Technology

From the list, select the type of DNA sequencer that you will use to analyze your target DNA fragments.

Sequencing Protocol

Select the desired protocol. In the list, eArray displays the available protocols for your chosen DNA sequencing technology.

Library Type

Select between creating a Normal library and a Supplemental library. A supplemental library is intended to be paired with an Agilent catalog library to create a combined library. Supplemental libraries can contain up to 57,750 baits, including the required Agilent quality control baits. A normal library is not paired with an Agilent catalog library, but if desired, it can later be used to create a combined library using the Combine Libraries wizard.

If you select Supplemental as the library type, you need to then make additional selections:

• Base Library Name drop-down list: Select the Agilent catalog library that you intend to later pair with the supplemental library you are creating. eArray sets certain Bait Tiling parameters for the supplemental library to those that were used to create the baits in the selected base library. Later, when you create a combined library that includes the selected base library and the supplemental library, the baits will be compatible.

• Ignore intervals already covered in base library check box: Mark the box if you want to skip adding baits to the supplemental library that overlap with genomic regions already covered by the chosen catalog library.

Design Options

Use Optimized Parameters

In general, mark Use Optimized Parameters. With this option, based on your selected sequencing technology and protocol, eArray automatically sets the best values for Design Strategy, Bait Length, Bait Tiling Frequency, and Allowed Overlap into Avoided Regions.

To set all of these options manually, clear Use Optimized Parameters. The options all become available.

• Note: When you create a supplemental library, you cannot clear this option.

Design Strategy

(Available if you clear Use Optimized Parameters.) In Design Strategy, select one of these options:

• Centered – eArray centers sets of tiled baits over their respective target intervals. Each set of baits hangs over both ends of its target interval by equal amounts. This option exactly respects the value that you select for Bait Tiling frequency.

• Justified – eArray designs sets of tiled baits to exactly cover their respective target intervals. This option adjusts the overlap of baits to achieve the precise and even tiling of only the target intervals, without any overhang into non-target regions. With this option, the actual tiling frequency can deviate from the value you select in Bait Tiling Frequency.

Bait Length

(Available if you clear Use Optimized Parameters.) Currently, eArray supports a bait length of 120 nucleotides.

• Note: All baits in a library must have the same length. If you intend to combine baits from two or more sources to create a library, make sure all baits have the same length.

Tiling Frequency

(Available if you clear Use Optimized Parameters.) Select the desired tiling frequency. This setting controls the density of tiling. The density can also be affected by your choice of Design Strategy. Not all bait tiling frequencies are available for every sequencing technology.

• 1x – eArray tiles baits end-to-end. All bases in each interval are covered by one bait.

• 2x – eArray overlaps baits by 50% as it tiles each interval. Two baits cover each base in each interval.

• 3x – eArray overlaps baits as it tiles each interval so that three baits cover each base in each interval.

• 4x – eArray overlaps baits as it tiles each interval so that four baits cover each base in each interval.

• 5x – eArray overlaps baits as it tiles each interval so that five baits cover each base in each interval.

• Note: Depending on your other settings, the exact density of tiling may be somewhat less than that indicated above at the extreme 5' and 3' ends of each interval.

Avoid Overlap

(Available if you clear Use Optimized Parameters.) To change the value, select it, then type the desired distance in base pairs. eArray generates baits that may extend by this distance into the regions specified in Genomic Avoid Intervals (see below). For best results, set this distance to a maximum of 20 bp.

Strand

Select one of these options:

• Sense – Creates baits in sense orientation. eArray selects this option by default.

• Antisense – Creates baits in antisense orientation.

• Both – Creates both sense and antisense baits.

Tiling Intervals

Enter Intervals

Enter the desired target genomic intervals, or upload them as a file.

To enter target intervals

1. Select Enter Intervals.

2. In the box, type the desired target intervals. Use the format chrX:<start>-<end>
   Example: chr21:1000000-1500000
   Separate multiple intervals with pipe "|" characters.

To upload intervals as a text or BED format file

1. Create or obtain a text or BED format file that contains the desired intervals.
   File format info

   File Type	Instructions/Details
   Plain text (*.txt)	Create a list of intervals as a *.txt file with one interval on each line. End each line with a new line character (in a word processor, press Enter at the end of each line). Use the format: chrX:<start>-<end> Example file content chr1:1234567-1678901 chr13:2000000-2500000 chrY:250000-975000
   BED (*.bed)	eArray supports the UCSC BED file format. Please note the following: The first three columns, in order, must be chromosome name, chromosome start, and chromosome end. Additional columns can be present after the first three required ones, but eArray ignores them. Do not include file or column headers. BED files are tab-delimited text files, with tab characters between items within a row, and a return character at the end of each row. For more information, see TDT format files. The file must have an extension of *.bed or *.BED Agilent recommends that you upload no more than 10,000 intervals at a time for use as Bait Tiling parameters. Example file content chr1     1234567     1678901 chr13    2000000     2500000 chrY     250000      975000

    

    

2. Select Upload Intervals.
   A new box appears.

3. Click Browse.
   A Choose File dialog box appears.

4. Select the desired file, then click Open.
   The name of the selected file appears in Genomic Target Intervals. eArray waits until after you submit the Bait Tiling job to upload the intervals from the file.

Extend Interval Boundaries 3' and 5'

eArray can tile baits into the regions that flank your entered genomic target intervals.

1. Mark Extend Interval Boundaries 3' and 5'.
   Two boxes appear.

2. In 5' bp and 3' bp, type the additional number of base pairs to be added to the 5' and 3' ends of each genomic target interval.

Avoid Standard Repeat Masked Regions

By default, eArray excludes a standard set of repetitive genomic regions from the Bait Tiling process. These are regions of the genome that generally produce poor quality baits. If you want to include repeat regions in the Bait Tiling process, clear this option.

Avoid Custom Intervals

eArray can exclude from the Bait Tiling process genomic intervals that you define. You can either enter the intervals, or upload a text file that contains them.

To enter custom exclusion intervals

1. Mark Avoid Defined Custom Intervals.
   A box appears.

2. In the box, type the desired intervals. Use the format chrX:<start>-<end>
   Example: chr21:1000000-1500000
   Separate multiple intervals with pipe "|" characters.

To upload a text file that contains custom exclusion intervals

1. Create a *.txt file with one interval per line. For each interval, use the format chrX:<start>-<end> (Example: chr21:1000000-1500000) End each line with a new line character (press Enter at the end of each line).

2. Mark Avoid Defined Custom Intervals.
   A box appears.

3. Next to the Avoid Defined Custom Intervals box, click Upload.
   A dialog box appears.

4. Click Browse.
   A Choose File dialog box appears.

5. Select the desired file, then click Open.
   The name of the file appears in the previous dialog box, in File Name.

6. Click Upload File.
   eArray uploads the list of intervals, and displays them in pipe-separated format in Avoid Defined Custom Intervals.

Statistic

Description

Total Target Regions

Number of base pairs in the entered genomic target regions for the Bait Tiling job.

Regions already covered in base library

Number of base pairs in the entered genomic target regions that are also covered in the selected base library. eArray will not design baits to these regions.

Regions not covered in base library

Number of base pairs in the entered genomic target intervals that are not also covered in the selected base library. eArray will design baits to these regions.

Parameter

Instructions/Details

Library Name

Type a name for the library. eArray uses this name as one of the search keys for libraries, and as a way to refer to it in search results, lists, and the like.

Base Library Name

(Read-only, supplemental libraries only) The name of the Agilent Catalog library (the "base library") that was selected for the bait tiling job. The new supplemental library can only be included with this specific Agilent Catalog library when you create a combined library.

Length

(Read-only) The bait length of the tiled baits (in nucleotides). All baits in a library are of the same length. Currently, eArray supports a bait length of 120 nucleotides.

Folder

Select a location for your new library. The folders to which you have access appear in the list.

Species

(Read-only) Reflects the species that you selected in the previous step of the wizard.

Library Size

(Read-only) Currently, eArray supports a library size of 1 x 55K, which can accommodate up to 57,750 baits, including the required Agilent quality control baits. Click Show Details to see additional information about available features and the control grid that will be applied. If you design a library that contains more than 57,750 baits, eArray creates a library set in which the requested baits are distributed over multiple libraries. See library sets.

Control Grid

(Read-only) eArray automatically selects an appropriate control grid for the library. The name of the Agilent control grid appears as a link. To view details about the control grid, click the link.

To select an alternate control grid, if one is available, click . A window opens where you can select the desired alternate control grid.

Agilent has two control grids: Hs and Generic. If you select H. sapiens as the species, eArray automatically selects the Hs control grid. Otherwise eArray selects the Generic control grid. These small groups do not affect your capture and are for Agilent QC purposes. For the bait groups that you add, generally users designate all of them with a control type of biological. If you have a control group planned (and you want to, for example, hybridize spiked-in DNA to those baits), then you could designate the group with a control type of Positive. This designation does not have any special effect on the synthesis of the library. Control baits are not listed in the BED file.

More Library Details (The properties below appear if you mark the More check box)

Description

(Optional) Type a brief description for the library.

Keywords

(Optional) Type search keywords to associate with the library, separated by commas. Keywords can help you search for the library.

Attachment

(Optional) Attachments are files or links that are related your library. Attached files can be up to 32 Mb in size. To add one or more attachments,  follow these steps:

1. Click .
   A page appears in a new window.

2. In the Attach pane, specify the following:

Name – Type a name for the attachment. This name becomes the link you click to access the attachment.

Type – Select the desired type of attachment from the list. An attachment can be either a file or a URL.

Locale – Select the appropriate locale from the list. eArray uses this option to select content that has been localized for your region, when it is available.

File – (Available only if you selected File for the type of attachment) Click Browse. Select the desired attachment in the dialog box that appears, then click Open.

URL – (Available only if you selected URL for the type of attachment) Type the full URL of the Internet resource, including the protocol specifier. Example: http://www.agilent.com.

3. Click Add.
   The attachment appears in the Attachment Header List at the top of the window. A success message appears.

4. Click Close.

5. Add additional attachments, if desired.
   If you add an attachment in error, select the check box next to its name in the Attachment Header List, then click Remove.

6. Click Done.
   The window closes. The names of your attachments appear as links in Attachment.

Comments

(Optional) Type comments to include with the library.

Task

Instructions/Details

Add bait
group(s)

(Available for Normal libraries, not Supplemental libraries) To add bait group(s) to your library:

1. Click Add.
   A bait group selection page appears.

2. In Bait Group Name, type all or part of the name of a bait group, then click Search. To return a list of all available bait groups, leave Bait Group Name empty.
   A list of bait groups appears.

3. Do any of the following to navigate through the list of bait groups.

   • To go to another page of results, click one of the Pages links.

   • To sort the list based on any column, click the heading of the desired column. To reverse the order of the items, click the column heading again.

   • To limit the list to a specific folder, select the desired folder in Folder, then click Filter.

4. Click the name of the desired bait group to select it. You can select more than one bait group on a page.

5. Click Add.
   eArray transfers the selected bait group(s) to the Selected Bait Group list.

6. Do any of the following to change the Selected Bait Group list:

1. • Add another bait group.

   • Do another bait group search. You can add bait groups from multiple searches.

   • Add all of the bait groups visible on a page — Click Add All.

   • Start over  — Click Remove All.

   • Remove a bait group — Select the desired bait group, then click Remove.

7. Click Done. You may need to scroll down to see this button.
   The selected bait groups appear under Biological SureSelect Target Enrichment Bait Group(s) in the main window of the wizard. To add additional bait groups to the library, click Add again.

View a bait group

In the Bait Group Name column, the names of bait groups in the library appear as links. Click the desired link to view details about the bait group.

The View Bait Group page appears in a new window. For details about this page, see View bait group.

Remove bait group

(Available for Normal libraries, not Supplemental libraries) To remove a bait group from the library:

1. Mark the check box next to the name of the bait group that you want to remove.

2. Click Remove.

Change number of copies of bait group

In the Replicate column, next to the desired bait group, type the number of copies of the bait group that you want to include in the library.

• Note:
  • eArray only lets you change the Replicate values for bait groups if you select Maximize capacity in Agilent Recommended Bait Boosting. Otherwise, eArray enforces a Replicate value of 1 for all bait groups in the library.
  
  • eArray replicates the required Agilent control bait group the same number of times as your least-replicated bait group.
  
  • eArray automatically includes as many complete sets of baits as will fit in the available space in the library. Example: Your library contains 12,000 baits, including the required Agilent control baits. The capacity of the 1 X 55K library format is 57,750 baits. eArray includes four complete sets of baits in your library (12,000 x 4 = 48,000 baits). Five complete sets would have exceeded the capacity of the library format (12,000 x 5 = 60,000 baits).

Change bait boosting preference

Bait boosting increases the representation of "orphan" and GC-rich baits within the library by an Agilent-defined factor. Agilent applies bait boosting to libraries by default.

More info about bait boosting

To change the bait boosting preference for the library

1. In Agilent Recommended Boosting, select one of these options:

   • Maximize Capacity – eArray applies no bait boosting to the library. This option lets you include the greatest number of user-defined baits in the library.

   • Balanced – eArray replicates any orphan or GC-rich baits present in your library by an Agilent-defined factor, potentially improving the library's capture of genomic fragments. This option uses a greater number of the available baits in the library than the Maximize Capacity option, but it uses fewer baits than the Maximize Performance option. Also, if you select this option, eArray enforces a Replicate value of 1 for all bait groups in the library.

   • Maximize Performance – To maximize the library's capture of genomic fragments, eArray replicates any orphan or GC-rich baits based on a sliding scale that reflects the boosting in Agilent’s catalog exome libraries. Baits with a higher GC content are replicated by a higher factor. This option can result in much higher levels of bait boosting than the Balanced option and, as a result, it uses the greatest number of the available baits in the library. Because the Maximize Performance option significantly boosts all GC-rich baits, Agilent does not recommend this option for libraries that cover non-coding regions with an atypically high GC content.

A dialog box asks if you are sure you want to change your boosting preference.

2. Click OK.
   Under Library Statistics, the value in Number of Available Features changes accordingly.