The functionality described in this topic is only available when you mark Show Advanced Options.

ChIP probegroup wizard:

Add Content

Select Probes from Agilent High Definition Database

 

Once you have chosen Select Probes from Agilent High Definition Database from the ChIP probegroup wizard: Add Content step, you will be directed to the following Define Targets step. Complete the fields and selections on this screen to define the desired genes or genomic regions that you want covered in the probegroup.

Screen 1 - Define Targets

Follow the steps below to define your targets.

  1. Enter the target identifiers in the Targets text box. You can type or paste the target identifiers directly into the text area, or click Upload to browse to a text file (*.txt) that lists the target identifiers (one identifier per line). See Permitted target identifiers for more information.

  2. Below the Databases heading, mark the genome databases that you want SureDesign to use to obtain genomic coordinate information for your specified targets. You can hover the cursor over a database name to see the date that Agilent most recently downloaded data from the database. The potential database sources are:

·        RefSeq - US National Center for Biotechnology Information (NCBI)

·        Ensembl - European Bioinformatics Institute and the Wellcome Trust Sanger Institute

·        CCDS - Consensus Coding Sequence project (CCDS) of the US National Center for Biotechnology Information (NCBI)

·        Gencode - US National Human Genome Research Institute (NHGRI) and the Wellcome Trust Sanger Institute

·        VEGA - Vertebrate Genome Annotation project of the Human and Vertebrate Analysis and Annotation (HAVANA) group at the Wellcome Trust Sanger Institute

·        CytoBand - CytoBand file from the UCSC Genome Browser

  1. In the Include Flanking Regions field, type the number of base pairs of flanking sequence (on the 3' and 5' ends) that you want SureDesign to include on each target. SureDesign does not include flanking bases for targets entered as genomic coordinates.

  2. Choose to select the Allow Synonyms check box, or leave de-selected. Allowing synonyms permits the use of synonym names to map the genes to a genomic location. In addition, if your target identifier is associated with more than one gene, the program will map the genomic location to two separate genes, and both genomic locations will be listed for the target. When the Allow Synonyms check box is cleared, SureDesign maps your targets to genomic locations using only the entered gene names. See Allow Synonyms for more information.

Click Next to continue to Screen 2.

Screen 2 - Review Targets

Review the Target Summary and Target Details to confirm that SureDesign successfully recognized all of the target identifiers entered on the previous screen.

·        The Target  Summary indicates the number of identifiers entered, found, and not found. If any of the target identifiers mapped to more than one genomic location, you will notice that the number of targets found is greater than the number of entered. See SureDesign gene finder for more information on how SureDesign maps target IDs to targets.

If SureDesign did not accurately identify all of your target regions

·        The Target Details table lists the Target ID you entered, the # Regions within the target, the number of Base Pairs within the regions defined by the target identifier, and the Position as genomic coordinates identified for the target.


Click Next to continue to Screen 3.

Screen 3 - Enter Parameters

Define parameters of your probegroup. Then, submit the design to SureDesign to begin the probe selection process.

Define probegroup parameters:

  1. Under Selection Method, use the radio buttons to indicate to SureDesign to select either a number of Total Probes for the probegroup, or a set number of Probes Per (target) Interval. Below these buttons, type your desired number of either total probes or probes per interval, depending on your selection. You must enter a number greater than 0.

  2. Use the Similarity Filter to set the specificity of the probes. Optimally, the HD probes that SureDesign selects hybridize to only one genomic location. Choose from:

·        No Filter - Select this option if you do not want SureDesign to filter probes based on specificity. Keep in mind that data from probes with more than one matching sequence in the genome may be harder to interpret.

·        Similarity Score - Select this option to filter out all probes with significant similarity to multiple genomic locations. This is the most stringent filter option. For some of your target regions, SureDesign may not be able to identify any probes in the HD database that pass this filter.

·        Perfect Match - Select this option to filter out all probes that match more than one location in the genome. If you select this option, SureDesign will not select any probes in segmental duplication regions or pseudo-autosomal regions.


Submit the probegroup for probe selection:

  1. Click Begin Probe Selection.

    A message box opens indicating the e-mail address where Agilent will contact you when the probe selection job is complete. If desired, you can enter additional e-mail addresses into the provided field.

  2. Click OK in the notification message to submit the probegroup to SureDesign.

    Your submission is placed in the SureDesign job queue to await probe selection. SureDesign searches the probes in Agilent's high definition (HD) probe database to locate the probes that cover your specified targets.

 

The wizard takes you to the Finalize step. Click Close to close the wizard.