Recalling Genotypes with BEAGLECALL Tutorial
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1 Recalling Genotypes with BEAGLECALL Tutorial Release Golden Helix, Inc. June 24, 2014
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3 Contents 1. Format and Confirm Data Quality 2 A. Exclude Non-Autosomal Markers B. Confirm Exact Matching of SNP Columns C. Confirm Matching Row Labels Using Scripts for Beagle Export and Import 5 A. BEAGLE Export Scripts B. BEAGLE Import Scripts i
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5 Recalling Genotypes with BEAGLECALL Tutorial, Release Updated: June 24th, 2014 Level: Advanced Packages: SNP Analysis, Power Seat BEAGLE and BEAGLECALL are Java-based tools for genetic analysis developed and maintained by Drs. Brian Browning and Sharon Browning of the University of Auckland. BEAGLE is particularly useful for inferring or imputing the genotypes of missing and untested SNPs. BEAGLECALL is a companion to BEAGLE that utilizes a combination of haplotype structure and allele intensity data to determine genotypes. This added data dimension results in very accurate genotype calls. This tutorial focuses on BEAGLECALL showcasing add-on scripts required for importing and exporting back and forth between SVS to BEAGLECALL. It also provides a workflow to maximize the efficiency of these products. Many of the scripts featured in this tutorial can also be used with BEAGLE, which has the same file formats as BEAGLECALL. Requirements To complete this tutorial you will need to download and unzip the following files, which includes several datasets. Download BEAGLE_Tutorial.zip BEAGLE_Files.zip Warning: The BEAGLE_Tutorial.zip file is large and may take awhile to download. Its main purpose is to help you follow along step-by-step. However, if you do not wish to download the file you can still use this tutorial as a general outline for your own study. We hope you enjoy the experience and look forward to your feedback. Contents 1
6 1. Format and Confirm Data Quality In order to run BEAGLECALL you need both pre-computed genotype calls and allele intensities as separate input files. Each major microarray platform provides different means for accessing this information. This tutorial provides a project that already contains both genotype calls and allele intensities from Affymetrix 6.0 data. For the purpose of your own study, you can find more details on obtaining this information from Affymetrix CHP and CEL files in the Affymetrix Files section of the SVS manual. If you have Illumina data, you can use the Golden Helix DSF Export plug-in for Genome Studio to generate both genotype calls and X/Y intensity values. See the plug-in s documentation for the details of this process. Make sure the X/Y intensity spreadsheet generated from the plug-in are oriented in the same manner as the A/B intensity spreadsheet in this project. A. Exclude Non-Autosomal Markers From the Project Navigator select File >Open Project and navigate to the project downloaded previously. This will open the project file described earlier. You should see the mapped genotype spreadsheet (Affy CHP Dataset - Mapped Sheet 1) and the mapped intensities spreadsheet (Affy CEL Dataset - Quantile Normalized - SNP Transposed - Mapped Sheet 1). Next you need to drop all unmapped and non-autosomal SNPs from the genotype and intensity spreadsheets. Open Affy CHP Dataset - Mapped Sheet 1. From the menu bar, choose Select >Activate by Chromosomes. Uncheck MT, X and Y so that only Chromosomes 1 through 22 are selected. Click OK. Then choose Select >Column >Column Subset Spreadsheet. Open Affy CEL Dataset - Quantile Normalized - SNP Transposed - Mapped Sheet 1. Inactive the nonautosomal chromosomes as before; Select >Activate by Chromosomes and uncheck MT, X and Y and click OK. Subset this spreadsheet as well; Select >Column >Column Subset Spreadsheet. B. Confirm Exact Matching of SNP Columns Intensities may be present for SNPs that failed initial calling, so it is necessary to only include SNPs that are present in both spreadsheets. Other scenarios for mismatches are also possible. First you will activate the columns in the intensities spreadsheet that have active genotype information, then you will reverse the order and confirm. Open the Affy CHP Dataset - Mapped Sheet 1 - Column Subset spreadsheet and choose Select >Apply Current Selection to Second Spreadsheet. Click Select Sheet and select Affy CEL Dataset - Quantile Normalized - SNP Transposed - Mapped Sheet 1 - Column Subset from the list and click OK. The dialog should look like Figure 1-1. Click OK. 2
7 Recalling Genotypes with BEAGLECALL Tutorial, Release Figure 1-1. Apply Filter to Spreadsheet dialog From Affy CEL Dataset - Quantile Normalized - SNP Transposed - Column Subset, choose Select >Subset Active Data. Right-click on the spreadsheet title in the bottom-left corner of the spreadsheet and choose Rename Node. Type Allele Intensities and click OK. From Allele Intensities choose Select >Apply Current Selection to Second Spreadsheet. Click Select Sheet and choose Affy CHP Dataset - Mapped Sheet 1 - Column Subset and click OK. The dialog should look like Figure 1-2. Click OK. Figure 1-2. Apply Filter to Spreadsheet dialog (Reverse direction) From Affy CHP Dataset - Mapped Sheet 1 - Column Subset choose Select >Subset Active Data. Rename the spreadsheet to Genotypes - matched. Confirm that both resulting spreadsheets have the same number of columns/snps (868,157 in this case). C. Confirm Matching Row Labels In order for the data to run correctly in BEAGLECALL, we need to make sure that the row labels match and are sorted exactly the same for both the genotypes and intensities spreadsheets. Open the two spreadsheets that were just edited and notice that in the Genotypes - matched spreadsheet the row labels have a.birdseed extension. Since the row labels need to match, use the Find and Replace tool available in the Spreadsheet Editor to edit all of the labels simultaneously. Open the Genotypes - matched spreadsheet and choose Edit >Edit this Spreadsheet. Right-click on the Sample column header above the row labels and choose Find and Replace... Type.birdseed in the Find what: box and leave the Replace with: box blank. The dialog should look like Figure 1-3. Click OK. C. Confirm Matching Row Labels 3
8 Recalling Genotypes with BEAGLECALL Tutorial, Release Figure 1-3. Find and Replace to remove.birdseed extension All of the row labels should now be red, meaning that they have been edited in the spreadsheet editor. Choose File >Save. Choose Current Spreadsheet after Create Spreadsheet as Child of. Click OK. You will also want to make sure that the row labels are in the same order for both spreadsheets. This step is very important because if the row labels for both spreadsheets are not in the same order, BEAGLECALL will fail to run. From Edited Genotypes - matched click on the empty cell above the Sample column header. This will sort the spreadsheet ascending. You should see and arrow pointing upwards in the right hand side of the box. From Allele Intensities click on the empty cell above the Columns column header. Both spreadsheets should now be sorted with ascending row labels Format and Confirm Data Quality
9 2. Using Scripts for Beagle Export and Import Now that the spreadsheets are properly formatted, you can use the BEAGLE/BEAGLECALL scripts to export the data. Before proceeding you will need to download the BEAGLE/BEAGLECALL scripts from the Add-on Scripts Repository, which have been bundled for ease of downloading. Put the export scripts in the...\userscripts\spreadsheet\file directory and put the import scripts in the...\user- Scripts\SVS\Import directory within your UserScripts folder. The easiest way to locate your UserScripts folder is to open the Project Navigator and choose Tools >Open Folder >User Scripts Folder. A. BEAGLE Export Scripts First use the scripts that export data from SVS in a way that will be properly formatted to run in BEAGLECALL. From the Edited Genotypes matched spreadsheet choose File >Export BEAGLECALL Genotype Probabilities. Choose an appropriate folder that you can find when running BEAGLECALL and click OK. This script will create 22.gprobs files containing the preliminary calls, one for each chromosome. Open the Allele Intensities spreadsheet. Choose File >Export BEAGLECALL Allele Signals. Again, choose an appropriate folder. This script will create 22.signals files. Warning: These export scripts can be time-consuming for large datasets. Your data should now be properly formatted to run BEAGLECALL. This tutorial does not lead you through running BEAGLECALL as we do not officially support it. For more information about installing and running BEAGLECALL on your system, please refer to the BEAGLECALL website. B. BEAGLE Import Scripts After running through the three iterations of BEAGLECALL as recommended in the BEAGLECALL manual, several files will be created. The ones we are interested in importing back into SVS are the gprobs and the phased files. If you were to use the entire genome, there would be 22 separate files of each type that need to be imported. If you do not wish to run BEAGLECALL right now, the necessary files for Chromosome 22 only are available for download, see Downloads under the Requirements section of this tutorial. They are named 5
10 Recalling Genotypes with BEAGLECALL Tutorial, Release bc3.chr22.chr22.signals.like.gz.gprobs.gz and bc3.chr22.chr22.signals.like.gz.phased.gz. The.gz extension means they are still compressed and need to be unzipped before you can import into SVS. After you have unzipped the files, they can be imported into SVS via the Import Beagle gprobs as Allelic Dosage and Import BEAGLE scripts. From the project navigator, choose Import >Import Beagle gprobs as Allelic Dosage. Navigate to the unzipped gprobs file. The gprobs file that was created after the third iteration of running BEA- GLECALL is named bc3.chr22.chr22.signals.like.gz.gprobs. Choose Whitespace Delimited after File Format and click OK. The resulting spreadsheet can be marker-mapped and used to perform numeric association tests or regression analysis. Use the Import BEAGLE script to import the phased file. From the project navigator, choose Import >Import BEAGLE. Again navigate to the unzipped.phased file, that is named bc3.chr22.chr22.signals.like.gz.phased. Select the file and click Open. The file will be imported as a genotype spreadsheet. Note: The import process does NOT preserve the genotype phase. Note: You may also wish to use the gprobs2beagle utility to create a beagle genotype file that has been filtered to meet a specified minimum probability (the phased output gives the most likely genotype for each position, even if the probability is low). The file created by gprobs2beagle can also be imported to SVS using the Import Beagle script. You should specify? as the missing-value character when running gprobs2beagle in order to ensure that the file is imported properly. You should now have an unmapped genotype spreadsheet with genotypic columns and samples as rows. After you have applied a maker map, you could perform a genotype association test. Please refer to our Genotype Association Testing section of our SNP Analysis tutorial for more information Using Scripts for Beagle Export and Import
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