tutorial for masterplex gt v2

Tutorial for MasterPlex® GT v2

© 2006 Hitachi Software Engineering America, Ltd. All Rights Reserved.

http://www.miraibio.com MasterPlex GT 2

The MasterPlex GT software is designed for genotyping analysis of Single Nucleotide Polymorphisms (SNPs) and Human Leukocyte Antigen (HLA) typing with data

generated from the Luminex system. In this tutorial, we will be going over five main

steps in analyzing a Luminex data file with the MasterPlex GT v2 software:

1) Using the Typing Table

2) Setting up Allele Call Parameters

3) Utilizing the Allele Call Window

4) Analyzing the Data with the Graph Window

5) Printing Reports

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Using the Typing Table

Let’s start by opening a sample SNP data

file. Press File and select Open Data File. Open the file called

“SampleBase.csv”. This should be

located in the following default directory: C:\Program Files\HitachiSoft\MasterPlex GT\SampleData

Displays the Percentage of total MFI for the Locus Displays the Bead Count

By default, the Typing Table will display the MFI values. The sample or well data are

displayed row by row. The loci or bead data are displayed column by column. To

display the Percentage of total MFI for each allele, click on the Show Percentage

icon. To display the Bead Count values, click on the Show Bead Count icon.

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View data by Locus

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Loci shown as tabs To view data by locus, press the

By Locus button and the loci will

appear as tabs. The data shown

only applies to the selected tab or

locus.

MasterPlex GT can calculate the

Average, Standard Deviation

(SD), and Coefficient of Variation in Percent (CV%) for the MFI, Adjusted MFI, and Count values

of each locus for a particular group

of samples. To do this, simply

select the samples you wish to

include in the calculation. To

select multiple samples, hold down

the Ctrl key and left-click the

samples. The calculations are

made immediately upon selection.


Gradient Background view Press the Gradient Background

button to view the intensity of the MFI

values. The darker the background

color, the higher the intensity or MFI

value.

Show Dendrogram

For Cluster Analysis, press the

Show Dendrogram icon. A

dendrogram will appear to the

left of the Well Names. By

default, Ward’s Method is used

to generate the dendrogram

based on Genotype. To

change the method or algorithm

of clustering the samples, simply

click on the desired method in

the Clustering Tool dialog box.

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Selecting Background Samples

Wells A1 and D3 are the background samples for this particular data file. To set the

background sample, right-click on the sample under Sample Name or Well Name and

select Local Negative Control. Repeat this for both samples and the average MFI of

each locus for these samples will be subtracted from all the other samples.

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Setting Up the Allele Call Parameters MasterPlex GT v2 allows the user to set the stringency of allele calling using three

different filters:

1) Minimum Events – Set the minimum Count. 2) Relative Intensity (RI) – Specify the minimum reportable RI. 3) Intensity – Specify the minimum reportable Median Fluorescence Intensity

(MFI).

Press this icon to open the

Parameter Setting window.

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The Parameter Setting window

allows the user to insert the Allele Call parameters:

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Minimum Events

Relative Intensity (RI) Intensity or (MFI)

The RI based Allele call uses two parameters: RI and Intensity Threshold.

The Intensity based Allele call just uses the Intensity Threshold.

For this tutorial, we will just use the default values of 20 Minimum Events, 25%

Minimum Reportable RI, and 35 Minimum Reportable MFI. To use these settings for all

loci, click on the Apply to all groups (loci) button. Since the organism for this data file

is diploid, please click on the Diploid radio button next to Ploidy and press the Apply this Ploidy to all groups (loci) button. The Call Parameters are now set for Allele Calling. Press the OK button to exit the Parameter Setting window.

Note: Bead counts lower than the minimum threshold will be highlighted in red in the

Bead Count view of the Typing Table.


Utilizing the Allele Call Window Press the Bell icon to view the Allele Calling results Paint Background toggle button

To view the results of the Allele Calling, press the Bell icon and the Allele Call window

will pop up. Just like the Typing Table, the samples are displayed by rows and the loci

are displayed by columns. The bases are highlighted by default if they are different

than the reference sample, which is A1 in this case. To highlight the bases that are

similar as compared to the reference sample, simply press the Paint Background

toggle button. To change the reference sample, click the radio button located to the left

of the sample name.

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To view the Allelic Frequencies,

click on the Allele Frequency tab on

the top. The count or frequency of

each allele for each locus will be

displayed along with the

corresponding percentages.

To view the Genotype Frequencies, press

the Genotype Frequency tab on the top.

The count is given for the appearance of

each genotype for each locus with the

corresponding percentages.


Close Window Haplotype Frequency tab

To view the Haplotype Frequency, simply click on the Haplotype Frequency tab. All

the Haplotypes with a frequency greater than or equal to 1 will be displayed along with

the total counts and percentages.

To close the Allele Call window, click on the X icon.

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Analyzing the Data with the Graph Window

MasterPlex GT enables the user to view the data in the form of bar graphs (2D and 3D), heatmaps, and scatter plots.

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There are two ways of opening the Graph

window:

Clicking on the Show Graph icon

Clicking on the Multi Graph link

The Graph window will initially be empty when it appears. To view the individual bar graphs for each sample, click on the corresponding samples under the Sample Name

table. To select multiple samples, hold down the Ctrl key and left-click. Please note

that the on-the-fly statistical calculations for the selected samples are also available

here on the left side of the screen.


Toggle 2D and 3D Button Toggle MFI and RI values Show Dendrogram

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By default, the graphs are all MFI based. To view graphs for the RI values, simple click

on the RI icon from the top icon menu. To switch back to the MFI based calculations,

click on the MFI icon.

To toggle between 2D and 3D views of the charts, press the Toggle 2D and 3D button

on the top icon menu.

The Show Dendrogram button is also available in the Graph window (Refer to Page 5

for more details on the Dendrogram feature).


Heatmap Depth Bar Graph Tab

The Heatmap is automatically displayed in the Graph window. It shows the intensity of

the MFI Percentage for each allele based on the Heatmap Color Indicator which goes

from black to red with red indicating the highest percentages. To view the locus names,

just hover over the each color panel with the mouse and a label will appear with the

locus name, MFI value, and Percentage of total MFI for each allele.

Press the Depth Bar Graph tab to view and compare all the selected samples in one

chart.

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MasterPlex GT offers two ways of analyzing and comparing a pair of samples or alleles

in the Graph window:

1) Sample by Sample – View the scatter plot of 2 selected samples

2) Allele by Allele – View the scatter plot of 2 selected alleles

Two-Sample Comparison Mode Sample by Sample Tab Correlation Coefficient

To begin comparison of two samples, press the Two-Sample Comparison Mode icon

and then click on the Sample by Sample tab. Next, select E1 and A2 from the Sample Name table and this will bring up the scatter plot with the Correlation Coefficient located on the top. The labels for each data point will appear if you click on the graph.

Here is a breakdown for the color coding of the data points:

White points – Alleles that are not called in either sample.

Red points – Alleles called in the Y-axis sample only.

Blue points – Alleles called in the X-axis sample only.

Black points – Alleles called in both samples.

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Allele by Allele Tab

To perform analysis of two alleles, press the Allele by Allele Tab. Next, select any two

alleles from the Allele Name table. This will bring up the scatter plot for the two alleles.

The labels for each data point will appear if you click on the graph. The color codes are

exactly the same as for Sample by Sample analysis:

White points – Alleles that are not called in either sample.

Red points – Alleles called in the Y-axis sample only.

Blue points – Alleles called in the X-axis sample only.

Black points – Alleles called in both samples.

Note: To Print Chart, Copy the Chart to a file, or Add To Report (Refer to the

Printing Reports section on page 19 for more details on this), just right-click and make

the desired selection.

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MasterPlex GT is capable of displaying the pairwise comparisons for all loci based on

correlation coefficients with the Homology Chart icon.

Show Homology Chart

The Homology Table gives the pairwise comparison of all loci based on the correlation

coefficients. The background color indicates the degree of similarity with blue signifying

100% identity.

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Homology Chart Tab

To view a 3D graphical representation of the Homology Table, press the Homology Chart tab. The y-axis represents the correlation coefficient from zero to one. The x-

axis and z-axis represent each locus in the order they are presented in the Homology Table under Well Name or Sample Name.

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Printing Reports MasterPlex GT allows the user to print the following reports:

Sample Information including Instrument Status, Background Information, and

Allele Call Parameter Settings

Allele Call Table – Allele, Genotype, and Haplotype Frequency Tables

All Charts created by the user

Cluster Analysis and Heatmap

Raw Data

Report Manager Icon

To launch the Report Manager, click on the Report Manager icon, which looks like a

camera. Choose the desired reports and press the Preview Report Button and the

Preview window will appear.

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Open Save Print Close

The Preview window enables the user to Open, Save, and Print reports. To close the

Preview window, click on the X icon.

Notes: To add a particular chart to the report, right-click on the chart and select Add to Report. To save your analysis data in MasterPlex GT, click on File and select Save Project. The project file may be opened later for further analysis by clicking on File and selecting

Open Project File.

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Congratulations! You have just finished analyzing the SNP data file utilizing MasterPlex

GT v2. As you can see, the MasterPlex GT Genotyping Software has been specifically

designed to simplify genotyping and haplotyping analyses from multiplexed data

generated with the xMAP platforms. Integrating a wide variety of graphical data

presentations as well as advanced statistics, MasterPlex GT provides rapid screening

and analysis for all genomic laboratories.


You can contact MiraiBio with any questions you have at (510) 337-2000 (USA phone number) or by sending an email to [email protected]. MiraiBio A Division of Hitachi Software 1201 Harbor Bay Parkway Suite 150 Alameda, CA 94502 Telephone 1.800.624.6176 1.510.337.2000 Facsimile 1.510.337.2099 Trademark Acknowledgments MasterPlex is a trademark of Hitachi Software Engineering Co., Ltd. Luminex® is a registered trademark of the Luminex Corporation. All other company and product names mentioned in this manual are trademarks or registered trademarks of their owners. © 2006 Hitachi Software Engineering America, Ltd. All Rights Reserved.

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