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Determining the average values for a region list

If you have a region list or a .BED file and you have a microarray experiment with data, you can summarize the microarray data according to by the genomic coordinates contained in the region list. For instanceexample, the region list contains a list of CpG islands, the experiment contains methylation percentage values for probes (β values), and you would like to summarize the methylation values for individual probes for the CpG islands. Or you have a list of copy number amplifications, microarray gene expression data, and you are interested in determining if the average intensities of the probes in those regions is higher than expected.

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Import the region list (or BED file) and specify the region property as explained elsewhere in this document

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of all probes in each CpG island.

• Import the region list (or .BED file) 

Be sure that you have added the region property. The list of region coordinates (chromosome, start, stop) from the region list will be mapped against the reference genome specified for the microarray data so specifying Species and Genome Build for your region list is unnecessary.  

• Open the microarray data spreadsheet

Samples should be on rows and data on columns in the microarray data spreadsheet. 

• Select the region list spreadsheet 
• Right-click any column header in the region list spreadsheet
• Select Insert Average from the pop-up menu (Figure 2)
  
  

If you have not specified an annotation source for the region list, a pop-up dialog will ask if you would like to add an association source. This is not necessary.

• If prompted, select No from the Missing annotation dialog
• Select the microarray data spreadsheet containing the values you want to average for each region from the Get average from spreadsheet drop-down menu

There are three options for averaging the data (Figure 3). Mean of samples significant in region is used when the region list has SampleIDs from the microarray data set associated with each region. In this case,

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Find region overlaps

You have a list of regions from another analysis program (perhaps you detected peaks using an R program) and you’d like to compare that region list with a region list that Genomics Suite calculated. Perhaps you have two lists created by Genomics Suite (one generated from peak detection with one set of parameters and the other created with different parameters) and you’d like to see what the two lists have in common. You may use the Tools > Find Region Overlaps command to compare two or more region lists as shown.

There are two separate modes of operation for this command: Report all regions and Only report regions present in all list. The first option, Report all regions, will report all regions in both lists. If there is any region overlap between the lists, the intersection of the regions will be reported along with the only the microarray data set samples specified for each region would be included in the mean calculation. Mean of all samples will add columns for the mean value of all probes for all samples and the number of probes for all samples in each region. Mean value for all samples separately will add two columns for each sample with the mean value of all probes for that sample and the number of probes for that sample in each region. 

• We have selected Mean value for all samples
• Select OK (Figure 3)
  
  

Columns will be added to the regions list spreadsheet. Here, we have added two columns with the average β-value for all samples in each CpG island and the number of probes in each CpG island (Figure 4). 

Find region overlaps

If you have two or more region lists with coordinates on the same reference genome, you can compare them to identify overlapping regions. 

• Open all region list spreadsheets that you want to compare 
• Select Tools from the main toolbar
• Select Find Region Overlaps (Figure 5)
  
  

The Find Region Overlaps tool has two modes of operation. The first, Report all regions, creates a new spreadsheet with any regions that did not intersect and all regions of intersection between any of the input lists. For each intersection, the start and stop coordinates of the intersection , and the percent overlap between the intersected region with each of the regions in the input lists . If a region is found in only one list, it will be reported as well.In contrast, the second option, are reported. The second, Only report regions present in all lists, will intersect both lists and only reports  creates a new spreadsheet with the intersected regions found in all the lists. 

Importing genomic locations to be used with annotating SNVs

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• Select your preferred mode; we have selected Only report regions present in all lists 
• Select Add New Spreadsheet to add any spreadsheets you want to compare; we are comparing two region list spreadsheets (Figure 6)
• Select OK 
  
  

A new region list spreadsheet will be created (Figure 7). The new region list is a temporary spreadsheet so be sure to save it if you want to keep it. 

Importing SNV genomic position list for annotation

To be annotated using the Annotate SNVs tool, an imported SNV position list must have four columns per locus: 

1. Position of the SNP listed as chr.basePosition
2. Sample ID or name
3. The reference base
4. The SNP call (sample genotype base)

• Prepare input list as shown in Figure 20 and save (Figure 8) with four columns describing the position, sample, reference base, and sample genotype base for each SNV 
  
  

• Save as either a tab-separated or comma separatedseparated filefile
• Use File > Import > Text to import the table. During import, change the data type of column 1 (as in Figure 3) to text by right-clicking on the color bar of column one and changing the data type to text. You may leave the other columns as categorical response types

• The correct properties must be set for this spreadsheet. Right-click on the newly imported spreadsheet in the navigator and select Properties

• Choose Other in the Configure Spreadsheet dialog (Figure 6)
• Make sure Genomic is selected in the Add Property pull-down menu and select Add In the next dialog box, select Genomic location instead of marker IDs in the Choose the type of genomic data. The Marker ID in column should be set to the first column. [If Marker ID in column does not contain any items in the pull-down list, it is likely that the first column was not a text column (drawn in gray) during import. If this happens, then right-click in the column header in the spreadsheet and change Type: to text.]

• Specify the Species from a pull-down menu selection or by typing in the species name. Select Edit Genome to specify the Species Name, Genome Version, Cytoband file, and 2Bit sequence file. The last two fields are optional. Select OK

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• the table as a text file
• Select Genomic data for What type of data is this file? 
• Set the position column Type to text 
• Set the other columns Type to categorical
• Select Genomic location instead of marker IDs from the Choose the type of genomic data drop-down menu of the Configure Genomic Properties dialog
• Specify the Species and Genome Build 
• Select OK

The Annotate SNVs tool can now be invoked on this spreadsheet. 

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