Agilent GeneSpringGT User Guide
GeneSpring GT (formerly Varia)
Quick Start Guide
The procedures in this guide are for use with Varia Analysis Workbench.
What is Varia Workbench?
Varia workbench provides tools for analyzing genetic variation on a
genomic scale. The program can analyze tens or hundreds of thousands
of genetic variations simultaneously. The program allows you to:
• View and navigate through a visual representation of all measured
variations in human chromosomes
• Perform genetic linkage and association tests to identify relationships
between genotypes and phenotypes
• View, calculate, and use haplotypes and haplotype maps
• View and analyze pedigrees
• Study the relationships between genetic variations, genes, and
sequence regions
What's New in Varia Workbench A.02.00
• Improved ease-of-use
• New analyses:
• Non-parametric linkage (NPL)
• ANOVA
• Regression
• Loss of Heterozygosity (LOH)
• Quantitative Case Control
• The ability to add variations and modify the genome
Agilent Technologies
Analyzing Data in Varia Workbench
This diagram shows the activities involved in analyzing data with Varia workbench.
For more information about
each analysis, see Tab le 1- 1
on page 3.
Note: Preparation steps shown
in shaded boxes are optional and
only apply to some analysis
types.
Import genotype data
Filter for variations
in experiment
Deduce haplotypes
See page 5
See page 6
See page 13
Import pedigree,
link individuals
to genotypes, and
import traits
Mendelian Inheritance
check; remove SNPs
and individuals with
Mendelian errors
• TDT
• HHRR
See page 7
See page 8
• ANOVA
• Case Control
• Regression
Association
(
See page 14)
Generate populations
(allele frequencies)
Select type of analysis
Linkage
See page 22)
(
Import pedigree,
link individuals
to genotypes, and
import traits
Mendelian Inheritance
check; remove SNPs
and individuals with
Mendelian errors
• Parametric Linkage
• Single Point NPL
• Multipoint NPL
See page 10
Other analyses
See page 26)
(
See page 7
See page 8
• Find Autozygous
Regions
• Loss of
Heterozygosity
2 Varia Analysis Workbench Quick Start Guide
Table 1-1 Types of Analyses in Varia Workbench
Analysis Type Pedigree? Description
Association Performed on larger outbred populations. (page 14)
ANOVA
(page 14)
Qualitative Case
Control (page 15)
Quantitative Case
Control (page 15)
Regression
(page 17)
Family-based
Association Analysis
(TDT and HHRR)
(page 19)
Linkage Performed on related groups of individuals. (page 22)
Parametric Linkage
(page 22)
Non-parametric
Linkage (NPL)
(page 24)
Other (page 26)
No Finds variations whose genotypes segregate individuals with respect to a quan-
titative trait. It is used when you have hundreds or thousands of unrelated individuals with high and low values of a quantitative trait.
No Estimates the linkage disequilibrium between two markers in a group of unre-
lated individuals with and without a particular phenotype.
No Estimates the linkage disequilibrium between two markers in a group of unre-
lated individuals with high and low values of a quantitative trait.
No Looks for association between genotypes and one or more phenotypes in a
large group of individuals (hundreds) when the non-genetic contributions to a
disease are thought to be important.
Yes Provides a complete set of linkage disequilibrium statistics that indicate the level
of association of a set of variations of interest to a specific trait or affliction
Requires a large number of individuals and pedigree data with known genotypes for both parents and their affected offspring.
Yes Estimates the recombination frequency between two markers when you have
genotyped many people in multiple generations in a pedigree.
Yes Localizes the genetic basis of a trait when you don’t have an exact model of the
genetics of the trait, but do know who is affected and unaffected by the disease
within a pedigree. A common example of NPL analysis is Affected Sibling Pair
analysis (ASP).
Find Autozygous
Regions
(page 26)
Loss of Heterozygosity (LOH)
(page 28)
No Finds homozygous regions in affected individuals that are likely to be inherited
from a common ancestor.
No Looks for statistically significant regions that are more homozygous in trans-
formed tissues than in pre-transformed tissues or in late-stage compared to
early-stage tumors.
Varia Analysis Workbench Quick Start Guide 3
Starting Varia Workbench
1 Install Varia workbench according to the instructions given in the Varia Workbench
Installation Manual.
2 Double-click the Varia workbench icon on the desktop.
3 A Helpful Hint is displayed that tells you how to load data. Click OK to close the
Hint window.
The Varia workbench main window appears.
Genome Browser
Navigator Pane
Colorbar
4 Proceed to “Import genotype data” on page 5.
Varia Workbench allows you to analyze data in many different ways. Often the
analyses are dictated by the data; how the study was conducted and whether or not
you have pedigree information for the individuals in the study.
4 Varia Analysis Workbench Quick Start Guide
Preparing for Analysis
The following activities are prerequisites for some analyses in Varia workbench:
Import genotype data
(Applies to all analyses.)
1 Select Import Data from the File menu.
2 Select a text file that contains the genotype data and click the Open button.
3 Confirm the selection of the correct data file format and genome on the Import
Data: Define File Format and Genome window, then click the Next button. In most
cases the file format will be Affymetrix or Silicon Genetics Internal SNP.
Verify that the variations that you are studying are in the current genome build. If not,
add your variations to the existing genome prior to importing data by using the Edit
Master Table of Variations command on the Edit menu. See Chapter 16 in the
User’s Guide for more information.
4 Add additional samples for experiment if needed on the Selected Files window, and
click the Next button.
5 (optional) Add attributes on the Sample Attributes window. Add any additional
information on the individuals in your experiment that is usually in the pedigree file
such as gender.
Note The Individual ID is the combined information from family identifier and individual
identifier in the pedigree file separated by a period (.). For example, if the first
individual in the pedigree comes from family 66 and has the individual identifier 1,
the Individual ID will be 66.1. You can add this now or from the Experiment
Checklist in Step 9.
6 (optional) Add an attribute field such as Founder, if you intend to generate the allele
frequencies using only the founders or any individual from your samples. The
founder-only method is recommended if you have more than 50 founders from the
same population in your sample set (Ott, 1992). Mark every individual that is a
founder with an uppercase F in the appropriate field so that you can sort on it later.
7 To add other attributes, copy the information from your pedigree file and paste it into
the respective attribute column. Make sure that the rows in the file you’ve copied
match the Sample Attribute window.
8 When you have finished adding attributes, click the Next button. You are prompted to
Create Experiment. Click the Ye s button to generate the experiment.
Varia Analysis Workbench Quick Start Guide 5
9 Name and save the experiment. Use the New Experiment Checklist to prepare your
data for analysis: Define Parameters, Define the Default Interpretation, and Link
Pedigree Individuals To Samples. It is also a good idea to assign a project to the
study at this point.
10 (optional) Modify attributes as described below.
Modify sample attributes
(Applies to all analyses.)
Use this procedure if you need to modify sample attributes.
1 Select Sample Manager from the Experiments menu.
2 Click the Filter on Experiment tab in the Sample Manager window.
3 Select your samples of interest in the Filter Results table.
4 Click the Add button. The samples appear in the Selected Samples table.
5 Click the Edit Attributes button to inspect, change, or add attributes. When you are
finished, click OK to close the Edit Attributes window.
6 Click OK to close the Sample Manager window.
Filter for variations in experiment
(Applies to all analyses.)
The Varia human genome contains millions of variations. Use the procedure below to
create a subset of these variations that contain data specific to your experiment.
1 Select Find Variations with Data in Experiment from the Tools menu.
2 In the Run Script window, select your experiment from the Experiments folder in
the Navigator pane and click the Experiment button in the Inputs area.
3 Click the Start button to run the script.
4Save the new variation list, and it will appear in the Variation Lists folder in the
Navigator pane of the Varia workbench main window.
5 (optional) Assign a project to the new variation list.
6 Varia Analysis Workbench Quick Start Guide
Import pedigree
(Applies to Linkage analyses and Family-based Association analyses only.)
A pedigree describing individuals, their relationships, and affliction status is required to
perform family-based genetic analysis. Use the procedure below to import a
tab-delimited pedigree text file. After the pedigree has been imported, match individuals
in the pedigree to corresponding genotyped individuals and define the traits that are
associated with the pedigree.
1 Select Import New Pedigree from the File menu.
2 Select the pedigree text file that corresponds to your experiment, then click the Open
button.
3 You are prompted to provide the meaning of any extra columns (traits).
4 Name and save the pedigree. It is also a good idea to assign a project to the pedigree
at this point.
5 A Link Individuals to Samples window opens automatically. Fill it out now or click
Cancel to close the window if you don’t want to fill it out at this time.
Link individuals to samples
Family-based analyses use genotyped individuals who have been matched to pedigree
individuals. When a pedigree is imported, the link window is automatically active, but
you can also open the window to make changes as described below.
a Right-click on the pedigree in the Navigator pane and select Inspect from the
shortcut menu to display the Pedigree Inspector.
b Click the Link to Experiment button on the Individuals in Pedigree tab to
display the Link Individuals to Samples window.
c Link each person from your pedigree to the experimental data. Each person in the
pedigree is listed in the Individuals table. The persons in the experiment file are
listed in the Samples table. Persons in the Individuals table are automatically
linked with persons in the Samples table if the Individual ID and Sample ID
match. These pairs are listed in the Individual/Samples Pairs table on the right
side of the Link Individuals to Samples window.
Alternate
method
Varia Analysis Workbench Quick Start Guide 7
You can also add Individual IDs to samples by selecting Deduce Pedigree from the
Tools menu.
Define traits
For some diseases, the inheritance model relating to a pedigree is known. The relevant
information such as phenotypes, penetrance, and allele frequencies can be defined for
these traits as described below.
a On the Trait Details tab of the Pedigree Inspector window, click the Add New
button.
b When the Edit Trait window opens, enter the values for the trait, define the
disease model, and add penetrance information.
c Click OK to close the Edit Trait window. The trait you just added appears in the
Trait Details tab of the Pedigree Inspector.
d Repeat Steps a-c for all traits you want to add.
Run Mendelian inheritance check
(Applies to Linkage analyses and Family-based Association analyses only.)
Prior to performing family-based parametric analyses, we recommend evaluating
variations for observance of Mendelian inheritance. This procedure will identify
genotyping errors.
1 Select Check Inheritance from the Tools menu.The Run Script window opens.
2 Select your experiment in the Navigator pane on the left side of the window, and
click the Genotypes button in the Inputs area.
3 Select the Vari a tio n Lis t for your experiment in the Navigator pane, and click the
To check button in the Inputs area. The variation list was created as described in
“Filter for variations in experiment” on page 6.
4 Select the Pedigree for your experiment in the Navigator pane, and click the
Pedigree button in the Inputs area.
5 Use the supplied default values for the following knobs, or enter different values if
you want:
• Allowable proportions of errors in %
• P value cutoff, to set what violates the inheritance rules
6 Click the Start button. The results are displayed in two tables; one that shows the
variations that seem to violate the inheritance rules and one that shows the
individuals that violate the inheritance rules.
7 Investigate the variations in the two lists. If necessary, variations or individuals with
unusually high genotyping errors can be removed as described below.
8 Varia Analysis Workbench Quick Start Guide
Remove individuals with Mendelian errors
Use this procedure after running the Mendelian inheritance check described on page 8.
a (optional) Make a copy of your existing pedigree as follows:
• Display the pedigree in Pedigree Diagram view in the Genome Browser pane of
the Varia workbench window.
• Select all the individuals in the pedigree, right-click in the Browser pane, and
select Make Pedigree from Selected Individuals from the shortcut menu.
• Name and save the copy of the pedigree.
b Using the Check-Inheritance Results table as a guide, remove the trios that have
inheritance errors as follows:
• Select the individuals in the Browser pane
• Right-click and select Delete Selected Individuals from the shortcut menu.
Note
If the parents have another child that does not show inheritance errors, then only remove
the child that shows errors, rather then the child and both parents.
c (optional) Re-run the Mendelian inheritance test to see if the variation list
changes.
Remove SNPs with Mendelian errors
Use this procedure after running the Mendelian inheritance check described on page 8.
a Select Physical Position from the View menu.
b Right-click on the variation list that contains the inheritance errors in Navigator,
and select Venn Diagram > Left from the shortcut menu.
c Right-click on the complete variation list for your experiment in Navigator (i.e.
the one that includes Mendelian errors), and select Venn Diagram > Right from
the shortcut menu.
d Select the complete variation list for your experiment again and set as Universe
using the Venn diagram option. Inspect the Venn diagram on the right side and
make sure the lists are correct.
e Right-click on the non-overlapping region in the complete variation list (the
green segment), and select Make list of variations in this list only from the
shortcut menu. The new variation list will contain only variations without
Mendelian errors.
Varia Analysis Workbench Quick Start Guide 9
Generate populations to establish allele frequencies
(Applies to some types of Linkage analyses and Autozygosity analyses.)
You can generate populations to establish allele frequencies in the following ways:
• Use your own samples (founders only) (page 10)
• Use Affymetrix frequencies (page 11)
• Import your own allele frequencies (page 11)
Use your own samples (founders only)
The following procedure allows you to generate a population containing allele
frequencies using individuals from your experiments.
1 Select Sample Manager from the Experiments menu.
a Click the Filter on Experiment tab, select the experiment of choice.
b Sort on attributes (e.g. Founders) in the Filter Results table.
c Click on each individual in the Filter Results table that you want to use to
generate allele frequencies. Click the Add button to add the samples to the
Selected Samples table. At least 50 founders should be selected.
d Click the Create Experiment button to create an experiment that contains the
individuals in the Selected Samples table.
2 Right-click on the sample file in the Experiment folder in the Navigator pane and
select Inspect from the shortcut menu.
a Click the Interpretation tab in the Experiment Inspector window.
b Click on Default Interpretation and select Do not Display.
c Save this file as a new interpretation (e.g. population condition). This results in a
single averaged condition with that interpretation.
3 Run the following script from the Basic Scripts folder of Navigator:
Merge-Split Groups > Merge Condition to Sample
a Select the single averaged condition that you generated in Step 2 from the
Experiment folder, and click the Condition button in the Inputs area.
b Leave the Data compression method as Population.
c Click the Start button to run the script. A new sample containing population allele
frequencies is created and displayed in a Sample Inspector window.
d Name and save the sample. It is also a good idea to assign a project to the sample
at this point. This sample is now available in the Sample Manager window.
10 Varia Analysis Workbench Quick Start Guide
4 (optional) Open the Sample Manager window from the Experiments menu.
a Sort the samples by date.
b Add the new population sample to the sample list.
c Create an experiment from this new sample.
Use Affymetrix frequencies
Affymetrix provides allele frequencies for their SNPs from African-American, Asian
and Caucasian populations. Contact Affymetrix for additional information.
1 Drag the Affymetrix Population Fre.zip file into the Varia workbench window, or
select Import Varia Zip from the File menu in the main window. Contact Agilent
Technical support for information on how to get this file if you don’t have it.
2 You can select these population files in the Experiments folder in the Navigator
pane.
Import your own allele frequencies
1 To import your own population frequencies, create an allele frequency file with the
following format. This is one of several input file formats supported in the Varia
Workbench.
# SiGSNP2.0 (Version)
# type=3 (Type of data: 3=population)
# chroms=2 (number of Chromosomes in organism)
# samples=1 (number of samples: 1 for populations)
# name=Population (identity of population)
rs1376173 A:0.2,G:0.8 (SNPID Allele1:Frequency1,Allele2:Frequency2)
rs1254601 C:0.2,T:0.8 (SNPID Allele1:Frequency1,Allele2:Frequency2)
rs1254600 C:0.8,T:0.2 (SNPID Allele1:Frequency1,Allele2:Frequency2)
Varia Analysis Workbench Quick Start Guide 11
2 Select Import Data from the File menu.
a Select the genome, such as Homo Sapiens build 34v2 db SNP.txt, and click the
Open button.
b Click the Next button on the Define File Format and Genome window.
c Add additional samples for experiment if needed on the Selected Files window,
and click the Next button.
d Click the Next button on the Sample Attributes window.
e You are prompted to Create Experiment. Click the Yes button to generate the
experiment.
fSave the experiment.
12 Varia Analysis Workbench Quick Start Guide
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