Bait Tiling creates baits that evenly span specified genomic regions of a given species. You can customize the tiling process for specific sequencing technologies and protocols, as well as select the length of baits and the density of tiling. If you like, you can use a wizard to guide you through the process of designing and submitting a bait library with Bait Tiling—see Create library by Bait Tiling (wizard). Additional details about bait tiling appear at the end of this topic under Additional Bait Tiling details.
Note: · Consider using Interval Finder or Exon Interval Finder to retrieve a list of genomic intervals to use as input for the Bait Tiling process. These tools find intervals of interest based on annotations that you specify. · For certain Agilent Catalog libraries, you can use Bait Tiling to add baits for additional genomic regions that are not otherwise represented. You first create a supplemental library that contains additional baits that are designed with the same parameters that were used for the selected Agilent Catalog library. For this purpose, be sure to use the special Create Supplemental Library by Bait Tiling wizard. You then create a combined library that contains the selected Agilent Catalog library and the supplemental library.
Click the Workspace tab. Bait Tiling is not available within a collaboration.
Set the application type to SureSelect Target Enrichment.
Click Baits > Bait Tiling. The Bait Tiling page appears.
Set the following parameters. All are required.
Parameter
Instructions/Details
Design Options
Design 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 Strategy
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.
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 only if you clear Use Optimized Parameters.) Select the desired bait length. All baits will be of exactly the length you select.
Note: · All baits in a library must be of 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. · For all users, eArray supports SureSelect Target Enrichment libraries with a bait length of 120 nucleotides. To enable libraries for your workgroup with bait lengths of 90, 150, and/or 170 nucleotides, contact Agilent Technical Support.
Bait Tiling Frequency
(Available only 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.
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 only 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.
Genome Build
(Read-only) This box shows the genome build of your selected species that eArray will use to generate baits.
Genomic Target Intervals
Type target intervals using the format chrX:<start>-<end> (for example, chr21:1000000-1500000). Separate multiple intervals with pipe "|" characters.
Alternatively, you can upload a list of intervals. Create the list as a *.txt file with one interval per line. End each line with a new line character — that is, in a word processor, press Enter at the end of each line. Then follow these steps:
Beside the Genomic Target Intervals box, click Upload. A dialog box appears.
Click Browse. A Choose File dialog box appears.
Select the desired file, then click Open. The name of the file appears in the previous dialog box, in File Name.
Click Upload File. eArray uploads the list of intervals, and displays them in pipe-separated format in the Genomic Target Intervals box.
Avoided Genomic Intervals
Mark any of these options:
Avoid Standard Repeat Masked Regions – 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. This option is marked by default.
Avoid Custom Intervals – In the Bait Tiling process, eArray excludes the genomic intervals that you specify in the box. Type intervals using the format chrX:<start>-<end> (for example, chr21:1000000-1500000). Separate multiple intervals with pipe "|" characters. Alternatively, you can upload a list of intervals. Create the list as a *.txt file with one interval per line. End each line with a new line character — that is, in a word processor, press Enter at the end of each line. Then follow these steps:
Beside the Avoid Custom Intervals box, click Upload. A dialog box appears.
Click Upload File. eArray uploads the list of intervals, and displays them in pipe-separated format in the Avoid Custom Intervals box.
Click Submit. eArray submits your Bait Tiling job to Agilent for processing. A message informs you that you will receive an e-mail when the job is complete.
Click Exit. The Bait Tiling page reappears. Your Bait Tiling job appears in the Search Results pane at the bottom of the page, where you can monitor its status. The list of jobs does not automatically update once it appears. Click Refresh to update the list to its most current form.
Status designations and what they mean
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.
It can take up to one day or more for eArray to finish your Bait Tiling job, depending on the number of jobs ahead of yours in the queue, and the size of your uploaded file. When eArray completes your Bait Tiling job, you receive an e-mail, and the job's status changes to Completed. You can then view or download the results, and you can also create a new bait group from them.
What design guidance can Agilent offer regarding target enrichment libraries?
When you generate custom SureSelect Target Enrichment Bait Libraries:
In Bait Tiling, avoid repeat regions with fairly high stringency (at most 20 bp overlap).
Follow the "Optimized" bait design strategy (Mark the Use Optimized Parameters check box). These parameters work well under standard conditions and are optimized for each given sequencing technology.
How much genomic space can be targeted?
Agilent can create libraries with up to 55,000 baits per kit. The default bait length is 120 nucleotides, and the default tiling frequency is 2X. Given these parameters, you can target up to 3.3 Mb. As you increase the tiling frequency, this limit decreases.
What is the largest region that can be targeted?
Assuming 120-mer baits and 2X tiling, the longest single region that can be captured is 3.3 Mb. However, you can identify regions for eArray to avoid in the tiling process as Avoided Genomic Intervals. These regions can include the default standard set of repeat regions, as well as custom regions that you specify. This can increase the size of the region that can be captured.
Why is 2X tiling helpful?
Baits tiled at a density of 2X cover target regions in a staggered manner. A target sequence that is represented at the end of a given bait is represented toward the middle of the alternate bait that covers the same region. For sequencing technologies that use end-sequencing, such as the Illumina technology, 2X tiling can help to sequence regions in the middle of a DNA segment more effectively.
If I have not reached the capacity of the library, should I increase the tiling frequency?
Yes. In general, the more tiling the better.
At a bait length setting of 120 nucleotides, what is the active capture size?
120 nucleotides.
What happens if a target region is shorter than the bait length?
eArray centers the resulting bait on the target region. The bait sequence includes enough of the immediately adjacent genomic regions to fill out the bait to its full, specified length.
Is DNA sequencing potential driven by bait length or by shear size?
Shear size. If a 120-mer bait pulls down a 500 bp DNA segment, the entire 500 bp segment is available for sequencing, without regard to the part that aligns with the 120-mer bait.
See Also
Baits
Target enrichment and eArray