6. Large-Scale Haplotype Association Testing

Now that you know how haplotype association testing works on a single-haplotype, perhaps you want to investigate haplotypes on a larger, multi-haplotype scale. For sake of computation time, this tutorial will lead you through haplotype association on chromosome 22 only, though the workflow can be applied directly to the entire genome.

A. Calculating Haplotype Blocks for Chromosome 22

Figure 7a. Block definitions spreadsheet.

• Open the HapMap Phenotype + 500K Genotypes spreadsheet and select Select >Activate by Chromosomes.
• Click Uncheck All and then check 22 and click OK.

This will create a new subset spreadsheet (HapMapPhenotype Dataset + HapMap 500K Genotypes - Sheet 2) where only genotypes in chromosome 22 are active.

• Rename the subset spreadsheet in the Project Navigator to, HapMap chr22.
• From HapMap chr22 select Analysis >Haplotype Block Detection.
• Keep the defaults and click Run.

A new block definition spreadsheet is created (Haplotype blocks, 1381 markers in 549 groups) with a single column representing various markers and the blocks they belong to (Figure 7a).

B. Haplotype Association Tests Using Block Definitions

• Open HapMap chr22 select Analysis >Haplotype Association Tests.
• Under Haplotype Block Definition select Use precomputed blocks.
• Click Choose Sheet. Select the Haplotype blocks, 1381 markers in 549 groups block definition spreadsheet and click OK.
• Keep the rest of the parameters the same as before (make sure Calculate per block is selected) and click Run.

A new p-value spreadsheet is created, Haplotype Association Tests (Per Block), this time with results for each haplotype block defined across chromosome 22.

• In the Project Navigator, rename this spreadsheet to Haplotype Association Tests (Per Block) - chr22.

C. Plotting Haplotype and Single Marker Association Results Together

To see if haplotypes provide additional power in association testing, you can compare haplotype association results side-by-side with single marker association results.

• Open the -log 10 P + LD plot and click on Graph 1 in the Graph Control Interface.
• Under the Add Item tab, click on the drop down menu and click Select Spreadsheet... Choose the Haplotype Association Tests (Per Block) - chr22 and click OK.
• Check Chi-Squared -log10 P and click Add.
• In the Graph Control Interface, rename the first Chi-Squared –log10 P graph item to Single Marker –log10 P (right-click >Rename) and the second to Haplotype –log10 P.
• Zoom out to chromosome 22 by double-clicking 22 in the Full Domain View (the top pane in the Plot Viewer Window).

Figure 7b. Single marker association vs. haplotype association results.

You can change the attributes of the Haplotype –log10 P graph item to differentiate it more from the Single Marker –log10 P graph item.

• Select the Haplotype –log10 P graph item and under the Item tab change the Line from No Line to Drop. Increase the line weight to 3 and the symbol size to 5. The result is shown in Figure 7b.

Notice only the single block defined earlier is displayed in the LD plot. You can add the automatically generated block set from the Graph Control Interface.

Figure 7c. Haplotype vs single marker association zoomed in.

• Select the Haplotype Block Set graph item in the Graph Control Interface and click Load….
• Select the Haplotype blocks, 1381 markers in 549 groups spreadsheet and click OK.

You now have a p-value plot with single marker and haplotype association results along with an LD plot of chromosome 22 with automatically defined haplotype blocks.

You can zoom in to any region by left-click and dragging in either graph or on a graph’s x-axis.

• Left-click on the p-valued plot’s x-axis and drag from one side of the significant peak in chromosome 22 to the other side (Figure 7c).