Create baits by Bait Tiling (SureSelect Target Enrichment)

Parameter

Instructions/Details

Job Info

Job Name

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

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.

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.

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. That is, each set of baits hangs over both ends of its target interval by equal amounts. This option exactly respects the value 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.

Bait 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. Availability of options for tiling density varies by sequencing technology.

• 1x – eArray tiles baits end-to-end. One bait covers each base in each interval.

• 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.

Allowed Overlap into Avoided Regions

(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.

Target Details

Species

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

Genomic Target Intervals

You can enter the desired target 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 Defined 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.

Status

Description

In Queue

The job has been submitted to Agilent, but no action has been taken on it yet.

Designing

The job has been submitted to Agilent, and processing has begun.

Completed

The job has been completed by Agilent, and the results are available for your use.

ERROR

The job has been submitted to Agilent, but there are problems. You must resubmit your job. eArray sends you an e-mail informing you of the error. This e-mail can contain additional information about the error.